Novel peptides and combination of peptides for use in immunotherapy against pancreatic cancer and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/409,393, filed May 10, 2019, which is acontinuation application of U.S. patent application Ser. No. 15/869,471,filed Jan. 12, 2018 (now U.S. Pat. No. 10,357,551, issued Jul. 23,2019), which is a continuation of U.S. patent application Ser. No.15/073,528 filed Mar. 17, 2016 (now U.S. Pat. No. 10,076,560, issuedSep. 18, 2018), which claims priority to U.S. Application No.62/134,253, filed Mar. 17, 2015, and GB 1504502.4, filed Mar. 17, 2015,the contents of which are incorporated herein by reference in theirentireties.

This application is related to PCT/EP2016/055817, filed Mar. 17, 2016,the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

A Sequence Listing is submitted herewith as an ASCII-compliant text filenamed “Sequence_Listing_2912919-041004_ST25”, created on Aug. 22, 2019,and having a size of 14,320 bytes as permitted under 37 C.F.R. §1.821(c). The material in the aforementioned file is hereby incorporatedby reference in its entirety.

BACKGROUND Field of the Invention

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

Description of Related Art

Pancreatic cancer is one of the most aggressive and deadly cancers inthe world. In 2012, it was the 12th most common cancer in men with178,000 cases and the 11^(th) most common cancer in women with 160,000cases worldwide. In the same year, 330,000 deaths were reported, makingpancreatic cancer the seventh most common cause of death from cancer(World Cancer Report, 2014).

Pancreatic cancer is not one single cancer entity, but several distinctsubtypes have to be distinguished. Exocrine tumors account forapproximately 95% of all pancreatic cancers and include ductal andacinary adenocarcinomas, intraductal papillary mucinous neoplasms(IPMN), solid pseudopapillary neoplasms, mucinous cystic adenomas andserous cystadenomas. The remaining 5% of all pancreatic cancers belongto the subgroup of pancreatic neuroendocrine tumors (World CancerReport, 2014).

Infiltrating ductal adenocarcinoma represents the most aggressive formof pancreatic cancer and due to its high frequency (90% of allpancreatic cancers), epidemiologic data mainly reflect this specificsubtype (World Cancer Report, 2014).

In 2012, 68% of all new cases occurred in developed countries, withhighest incidence rates in central and Eastern Europe, North America,Argentina, Uruguay and Australia. In contrast, most countries in Africaand East Asia display low incidence rates. Globally, incidence ratesappear to be rather stable over time in both genders (World CancerReport, 2014).

Due to a lack of specific symptoms, pancreatic cancer is typicallydiagnosed at an advanced and often already metastatic stage. Theprognosis upon diagnosis is very poor, with a 5 years survival rate of5% and a mortality-to-incidence ratio of 0.98 (World Cancer Report,2014).

Several factors have been reported to increase the risk to developpancreatic cancer, including older age, as most patients are older than65 years at diagnosis, and race, as in the USA the Black population hasan 1.5 fold increased risk compared to the White population. Furtherrisk factors are cigarette smoking, body fatness, diabetes, non-0 AB0blood type, pancreatitis and a familial history of pancreatic cancer(World Cancer Report, 2014).

Up to 10% of all pancreatic cancer cases are thought to have a familialbasis. Germline mutations in the following genes are associated with anincreased risk to develop pancreatic cancer: p16/CDKN2A, BRCA2, PALB2,PRSS1, STK11, ATM and DNA mismatch repair genes. Additionally, thesporadic cases of pancreatic cancer are also characterized by mutationsin different oncogenes and tumor suppressor genes. The most commonmutations in ductal adenocarcinoma occur within the oncogenes KRAS (95%)and AIB1 (up to 60%) and the tumor suppressor genes TP53 (75%),p16/CDKN2A (95%) and SMAD4 (55%) (World Cancer Report, 2014).

Therapeutic options for pancreatic cancer patients are very limited. Onemajor problem for effective treatment is the typically advanced tumorstage at diagnosis. Additionally, pancreatic cancer is rather resistantto chemotherapeutics, which might be caused by the dense andhypovascular desmoplastic tumor stroma.

According to the guidelines released by the German Cancer Society, theGerman Cancer Aid and the Association of the Scientific MedicalSocieties in Germany, resection of the tumor is the only availablecurative treatment option. Resection is recommended if the tumor isrestricted to the pancreas or if metastases are limited to adjacentorgans. Resection is not recommended if the tumor has spread to distantsites. Resection is followed by adjuvant chemotherapy with gemcitabineor 5-fluorouracil +/− leucovorin for six months (S3-Leitlinie ExokrinesPankreaskarzinom, 2013).

Patients with inoperable tumors in advanced stage can be treated with acombination of chemotherapy with radiation-chemotherapy (S3-LeitlinieExokrines Pankreaskarzinom, 2013).

The standard regimen for palliative chemotherapy is gemcitabine, eitheras monotherapy or in combination with the EGF receptor tyrosine kinaseinhibitor erlotinib. Alternative options are a combination of5-fluorouracil, leucovorin, irinotecan and oxaliplatin, also known asFOLFIRINOX protocol or the combination of gemcitabine withnab-paclitaxel, which was shown to have superior effects compared togemcitabine monotherapy in the MPACT study (Von Hoff et al., 2013;S3-Leitlinie Exokrines Pankreaskarzinom, 2013).

The high mortality-to-incidence ratio reflects the urgent need toimplement more effective therapeutic strategies in pancreatic cancer.

Targeted therapies, which have already been shown to be efficient inseveral other cancer entities, represent an interesting option.Therefore, several studies have been performed to evaluate the benefitof targeted therapies in advanced pancreatic cancers, unfortunately withvery limited success (Walker and Ko, 2014). Nevertheless, the geneticdiversity of pancreatic cancer might offer the possibility ofpersonalized therapy, as invasive ductal adenocarcinoma with biallelicinactivation of BRCA2 or PALB2 was shown to be more sensitive to poly(ADP-ribose) polymerase inhibitors and mitomycin C treatment (WorldCancer Report, 2014).

Targeting the tumor stroma constitutes an alternative approach todevelop new treatments for pancreatic cancer. The typically dense andhypovascular stroma might function as barrier for chemotherapeutics andwas shown to deliver signals that promote tumor proliferation, invasionand cancer stem cell maintenance. Thus, different preclinical andclinical studies were designed to analyze the effect of stromaldepletion and inactivation (Rucki and Zheng, 2014).

Vaccination strategies are investigated as further innovative andpromising alternative for the treatment of pancreatic cancer.Peptide-based vaccines targeting KRAS mutations, reactive telomerase,gastrin, survivin, CEA and MUC1 have already been evaluated in clinicaltrials, partially with promising results. Furthermore, clinical trialsfor dendritic cell-based vaccines, allogeneic GM-CSF-secreting vaccinesand algenpantucel-L in pancreatic cancer patients also revealedbeneficial effects of immunotherapy. Additional clinical trials furtherinvestigating the efficiency of different vaccination protocols arecurrently ongoing (Salman et al., 2013).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and pancreatic cancer in particular.There is also a need to identify factors representing biomarkers forcancer in general and pancreatic cancer in particular, leading to betterdiagnosis of cancer, assessment of prognosis, and prediction oftreatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 67 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 67, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 67 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 67,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1I, 2A-2C, and 3A-3D depict embodiments according to thepresent disclosure.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4 and 4-2 are furthermore useful inthe diagnosis and/or treatment of various other malignancies thatinvolve an over-expression or over-presentation of the respectiveunderlying polypeptide.

TABLE 1 Peptides according to the present invention SEQ ID No. SequenceGeneID (s) Official Gene Symbol (s)  1 FLAQQESEI 1211, 1212 CLTA, CLTB 2 SLQEEHVAVA 5339 PLEC  3 ALLTFMEQV 165 AEBP1  4 SVDVSPPKV 113146AHNAK2  5 LLVDDSFLHTV 253982 ASPHD1  6 VLISLKQAPLV 1211 CLTA  7AQQESEIAGI 1211, 1212 CLTA, CLTB  8 IVDDLTINL 1303 COL12A1  9 FLFDGSANLV1293 COL6A3 10 FLVDGSSAL 1293 COL6A3 11 FLYKIIDEL 1293 COL6A3 12FVSEIVDTV 1293 COL6A3 13 LLAGQTYHV 1293 COL6A3 14 VLAKPGVISV 1293 COL6A315 SLANNVTSV 131566 DCBLD2 16 APVNVTTEVKSV 158078, 1915 EEF1A1P5, EEF1A117 FLKSGDAAIV 158078, 1915 EEF1A1P5, EEF1A1 18 SLLDDELMSL 26088 GGA1 19HLAPETDEDDL 8100 IFT88 20 RLAGDGVGAV 3855 KRT7 21 HLMDQPLSV 3918 LAMC222 TLDGAAVNQV 3918 LAMC2 23 SLSAFTLFL 4060 LUM 24 GLLEELVTV 642475 MROH625 SLKEEVGEEAI 4627 MYH9 26 SLKEEVGEEAIV 4627 MYH9 27 YLQGQRLDNV 6447SCG5 28 YLQGQRLDNVV 6447 SCG5 29 FLQEYLDAI 6317, 6318 SERPINB3, SERPINB430 VVDEGPTGV 9123 SLC16A3 31 SLAAAAGKQEL 6750 SST 32 SLAAAAGKQELA 6750SST 33 SLDSRLELA 81628 TSC22D4 34 MLMPVHFLL 114131 UCN3 35 VMDSGDGVTHTV100996820, ACTBL2, POTEKP, 344227, 345651, POTEE, ACTB, POTEM,440915, 445582, POTEI, POTEJ, ACTG1, 60, 641455, POTEF 653269, 653781,71, 728378 36 KQEYDESGPSIVH 100996820, POTEKP, POTEE, 344227, 440915,ACTB, POTEM, POTEI, 445582, 60, POTEJ, ACTG1, POTEF 641455, 653269,653781, 71, 728378 37 GLLKKINSV 55107 ANO1 38 NLVEKTPALV 10632, 267020ATP5L, ATP5L2 39 TLLSNLEEA 1191 CLU 40 FILDSAETTTL 1293 COL6A3 41FLLDGSEGV 1293 COL6A3 42 KLVDKSTEL 1293 COL6A3 43 RLDQRVPQI 1293 COL6A344 VLLDKIKNLQV 1293 COL6A3 45 VADKIHSV 11072 DUSP14 46 TFAPVNVTTEVKSV158078, 1915 EEF1A1P5, EEF1A1 47 KMDASLGNLFA 10447, 51384 FAM3C, WNT1648 ALTQTGGPHV 2316 FLNA 49 NLKGTFATL 100187828, 3043, HBB, HBD 3045 50ALAAILTRL 80201 HKDC1 51 ALMLQGVDL 3329 HSPD1 52 RMVEEIGVEL 10525 HYOU153 SSFGGLGGGSV 3880 KRT19 54 VLLSEIEVA 4134 MAP4 55 YLDAMMNEA103910, 10627 MYL12B, MYL12A 56 GLLDYATGAIGSV 117583 PARD3B 57 FLGKVVIDV100271927, 10156 RASA4B, RASA4 58 GLAAFKAFL 5999 RGS4 59 KLFNLSKEDDV6194 RPS6 60 YLEEDVYQL 23255 SOGA2 61 ALEKDYEEVGV 10376, 113457,TUBA1B, TUBA3D, 7278, 7846 TUBA3C, TUBA1A 62 ALEKDYEEV 10376, 113457,TUBA1B, TUBA3D, 51807, 7277, 7278, TUBA8, TUBA4A, 7846, 84790TUBA3C, TUBA1A, TUBA1C 63 FAGDDAPR 100996820, POTEE, ACTA1,344227, 445582, ACTA2, ACTB, POTEI, 58, 59, 60, 653269, POTEJ, ACTC1,653781, 70, 71, 72, ACTG1, ACTG2, POTEF 728378 64 FLVSNMLLAEA 113791PIK3IP1

TABLE 2 Additional peptides according to the presentinvention with no prior known cancer association SEQ ID Official No.Sequence GeneID (s) Gene Symbol (s) 65 YLYDSETKNA 4316 MMP7 66 ALLSGLREA23028 KDM1A 67 KMFFLIDKV 4599 MX1

TABLE 3 Peptides useful for e.g. personalized cancer therapies SEQ IDOfficial No. Sequence GeneID (s) Gene Symbol (s) 68 KLLTEVHAA 101 ADAM869 VMAPFTMTI 338 APOB 70 FLVDGSWSV 1303 COL12A1 71 FLLDGSANV 1293 COL6A372 YVYQNNIYL 2191 FAP 73 TLVAIVVGV 60681 FKBP10 74 KIQEILTQV 10643IGF2BP3 75 RLDDLKMTV 3918 LAMC2 76 RLLDSVSRL 3918 LAMC2 77 GLTDNIHLV25878 MXRA5 78 TLSSIKVEV 25878 MXRA5 79 VLAPRVLRA 5954 RCN1 80 TLYPHTSQV1462 VCAN 81 AMSSKFFLV 7474 WNT5A 82 SISDVIAQV 56172 ANKH 83 FLIDSSEGV1293 COL6A3 84 NLLDLDYEL 1293 COL6A3 85 TVAEVIQSV 55083 KIF26B 86SLLAQNTSWL 7070 THY1 L 87 LLLGSPAAA 23544 SEZ6L

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, lung cancer, kidneycancer, brain cancer, colon or rectal cancer, esophageal cancer, breastcancer, ovarian cancer, stomach cancer, liver cancer, prostate cancer,melanoma and leukemias.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 67. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 34 (see Table 1), and their uses in theimmunotherapy of pancreatic cancer, lung cancer, kidney cancer, braincancer, colon or rectal cancer, esophageal cancer, breast cancer,ovarian cancer, stomach cancer, liver cancer, prostate cancer, melanomaand leukemias, and preferably pancreatic cancer. As shown in thefollowing Table 4 and 4-2, many of the peptides according to the presentinvention are also found on other tumor types and can, thus, also beused in the immunotherapy of other indications. Also refer to FIGS.1A-1I and Example 1.

TABLE 4Peptides according to the present invention and their specific uses in otherproliferative diseases, especially in other cancerous diseases. The table showsfor selected peptides on which additional tumor types they were found and over-presented on at least 5% of the measured tumor samples, or presented on more than5% of the measured tumor samples with a ratio of geometric means tumor vs normaltissues being larger than 3. Over-presentation is defined as higher presentationon the tumor sample as compared to the normal sample with highest presentation.SEQ ID No. Sequence Other relevant organs/diseases  3 ALLTFMEQVLung, Kidney, Brain, Colon, Rectum, Esophagus  4 SVDVSPPKVLung, Kidney, Melanoma  5 LLVDDSFLHTVKidney, Brain, Liver, Melanoma, Ovary  8 IVDDLTINL Esophagus  9FLFDGSANLV Lung, Colon, Rectum, Breast, Esophagus 10 FLVDGSSALLung, Stomach, Breast 11 FLYKIIDEL Lung, Colon, Rectum, Breast 12FVSEIVDTV Lung, Breast, Esophagus 14 VLAKPGVISV Lung 15 SLANNVTSVLung, Kidney, Brain, Stomach, Melanoma, Ovary, Esophagus 16 APVNVTTEVKSVLeukocytes 21 HLMDQPLSV Lung 23 SLSAFTLFL Lung, Prostate 24 GLLEELVTVLung, Stomach, Ovary 30 VVDEGPTGV Lung, Kidney, Brain, Stomach, Liver,Leukocytes, Breast, Ovary 34 MLMPVHFLL Stomach 36 KQEYDESGPSIVHLung, Brain 39 TLLSNLEEA Brain, Prostate 40 FILDSAETTTL Lung 41FLLDGSEGV Lung, Breast, Ovary, Esophagus 42 KLVDKSTELLung, Colon, Rectum, Esophagus 43 RLDQRVPQILung, Colon, Rectum, Breast, Esophagus 44 VLLDKIKNLQVLung, Stomach, Colon, Rectum, Liver, Breast, Melanoma 45 VADKIHSVKidney, Stomach 47 KMDASLGNLFA Brain 50 ALAAILTRLKidney, Stomach, Colon, Rectum 51 ALMLQGVDL Esophagus 53 SSFGGLGGGSVLung 55 YLDAMMNEA Brain, Colon, Rectum, Liver, Ovary 58 GLAAFKAFLLung, Kidney, Liver 60 YLEEDVYQL Lung, Kidney, Colon, Rectum, Breast 64FLVSNMLLAEA Prostate 65 YLYDSETKNA Kidney, Colon, Rectum, Liver, Ovary,Esophagus 66 ALLSGLREA Kidney, Leukocytes, Melanoma 67 KMFFLIDKVBrain, Liver 68 KLLTEVHAA Lung, Kidney, Stomach, Colon, Rectum,Liver, Breast, Ovary 69 VMAPFTMTILung, Liver, Prostate, Ovary, Esophagus 70 FLVDGSWSVLung, Stomach, Colon, Rectum, Ovary, Esophagus 71 FLLDGSANVLung, Stomach, Colon, Rectum, Liver, Breast, Ovary, Esophagus 72YVYQNNIYL Lung, Stomach, Colon, Rectum, Liver,Breast, Melanoma, Ovary, Esophagus 73 TLVAIVVGVLung, Kidney, Brain, Stomach, Colon,Rectum, Liver, Prostate, Breast, Ovary, Esophagus 74 KIQEILTQVLung, Kidney, Brain, Stomach, Colon, Rectum, Liver, Leukocytes, Ovary,Esophagus 75 RLDDLKMTV Lung, Kidney, Colon, Rectum, Ovary, Esophagus 76RLLDSVSRL Lung, Kidney, Colon, Rectum, Liver, Ovary 77 GLTDNIHLVLung, Kidney, Colon, Rectum, Ovary, Esophagus 78 TLSSIKVEVLung, Kidney, Stomach, Colon, Rectum, Prostate, Breast, Melanoma 79VLAPRVLRA Lung, Kidney, Brain, Colon, Rectum, Liver 81 AMSSKFFLVLung, Brain, Stomach, Colon, Rectum, Liver, Prostate, Esophagus 82SISDVIAQV Lung, Brain, Colon, Rectum, Liver, Prostate 83 FLIDSSEGVLung, Colon, Rectum, Breast, Ovary, Esophagus 84 NLLDLDYELLung, Stomach, Colon, Rectum, Breast, Ovary, Esophagus 85 TVAEVIQSVLung, Esophagus 86 SLLAQNTSWLL Lung, Kidney, Brain, Stomach, Colon,Rectum, Liver, Melanoma 87 LLLGSPAAA Brain

TABLE 4-2Peptides according to the present invention and their specific uses in other proliferative diseases, especially in other cancerous diseases (amendment ofTable 4). The table shows, like Table 4, for selected peptides on whichadditional tumor types they were found showing over-presentation (includingspecific presentation) on more than 5% of the measured tumor samples, orpresentation on more than 5% of the measured tumor samples with a ratio ofgeometric means tumor vs normal tissues being larger than 3. Over-presentationis defined as higher presentation on the tumor sample as compared to the normalsample with highest presentation. Normal tissues against which over-presentationwas tested were: adipose tissue, adrenal gland, blood cells, blood vessel, bonemarrow, brain, cartilage, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung, lymph node, nerve, pancreas, parathyroid gland, peritoneum, pituitary,pleura, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach,thymus, thyroid gland, trachea, ureter, and urinary bladder.  SEQ ID No.Sequence Additional Entities  3 ALLTFMEQVSCLC, BRCA, Melanoma, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer  4 SVDVSPPKVMelanoma, Esophageal Cancer  5 LLVDDSFLHTSCLC, BRCA, Melanoma, Esophageal Cancer, Uterine VCancer, Gallbladder Cancer, Bile Duct Cancer  6 VLISLKQAPLVBRCA, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer  8IVDDLTINL NSCLC, GC, Melanoma, Uterine Cancer, GallbladderCancer, Bile Duct Cancer, NHL  9 FLFDGSANLVSCLC, Melanoma, OC, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer 10 FLVDGSSALSCLC, CRC, Melanoma, Esophageal Cancer, Urinarybladder cancer, Gallbladder Cancer, Bile Duct Cancer 11 FLYKIIDELSCLC, Melanoma, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer 12 FVSEIVDTVSCLC, GC, CRC, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer 13 LLAGQTYHVNSCLC, BRCA, OC, Esophageal Cancer, Urinary bladdercancer, Gallbladder Cancer, Bile Duct Cancer 14 VLAKPGVISVBRCA, Gallbladder Cancer, Bile Duct Cancer 15 SLANNVTSVUrinary bladder cancer, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer 16 APVNVTTEVK AML SV 19 HLAPETDEDDGallbladder Cancer, Bile Duct Cancer L 20 RLAGDGVGAUrinary bladder cancer V 21 HLMDQPLSVOC, Esophageal Cancer, Uterine Cancer, GallbladderCancer, Bile Duct Cancer 22 TLDGAAVNQVEsophageal Cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer23 SLSAFTLFL SCLC, BRCA, Melanoma, OC, Esophageal Cancer, Urinarybladder cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 24 GLLEELVTVSCLC, CRC, BRCA, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 29FLQEYLDAI Urinary bladder cancer 30 VVDEGPTGVSCLC, CRC, Melanoma, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, NHL 34 MLMPVHFLL BRCA 37GLLKKINSV BRCA, Esophageal Cancer, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer, OC 38 NLVEKTPALV AML 39 TLLSNLEEAUrinary bladder cancer, Uterine Cancer, NHL 40 FILDSAETTTLSCLC, BRCA, OC, Esophageal Cancer 41 FLLDGSEGVSCLC, Melanoma, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer 42 KLVDKSTELSCLC, BRCA, Melanoma, Gallbladder Cancer, Bile Duct Cancer 43 RLDQRVPQISCLC, Gallbladder Cancer, Bile Duct Cancer 44 VLLDKIKNLQVSCLC, OC, Esophageal Cancer, Urinary bladder cancer,Gallbladder Cancer, Bile Duct Cancer, NHL 45 VADKIHSVBRCA, Melanoma, Esophageal Cancer, Urinary bladder cancer 46 TFAPVNVTTEGallbladder Cancer, Bile Duct Cancer VKSV 47 KMDASLGNLFEsophageal Cancer, Urinary bladder cancer A 50 ALAAILTRLUterine Cancer, Gallbladder Cancer, Bile Duct Cancer 51 ALMLQGVDL BRCA53 SSFGGLGGG BRCA SV 54 VLLSEIEVA Melanoma, Uterine Cancer 55 YLDAMMNEAPrC, Melanoma, Urinary bladder cancer, Gallbladder Cancer,Bile Duct Cancer 58 GLAAFKAFLSCLC, BRCA, Melanoma, OC, Esophageal Cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, NHL, OC 60 YLEEDVYQLMelanoma, Esophageal Cancer, Urinary bladder cancer,Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer, NHL 64 FLVSNMLLAEUrinary bladder cancer A 65 YLYDSETKNASCLC, BRCA, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 66ALLSGLREA GC, BRCA 67 KMFFLIDKVBRCA, Melanoma, OC, Urinary bladder cancer, UterineCancer, Gallbladder Cancer, Bile Duct Cancer, NHL, OC NSCLC = non-smallcell lung cancer, SCLC = small cell lung cancer, RCC = kidney cancer,CRC = colon or rectum cancer, GC = stomach cancer, HCC = liver cancer,PC = pancreatic cancer, PrC = prostate cancer, BRCA = breast cancer, MCC= Merkel cell carcinoma, OC = ovarian cancer, NHL = non-Hodgkinlymphoma, AML = acute myeloid leukemia, CLL = chronic lymphocyticleukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 3, 4, 9, 10, 11, 12, 14, 15, 21, 23, 24, 30, 36, 40, 41, 42, 43,44, 50, 53, 58, 60, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81,82, 83, 84 85, and 86 for the—in one preferred embodimentcombined—treatment of lung cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 3, 4, 5, 15, 30, 45, 50, 58, 60, 65, 66, 68, 73, 74, 75, 76, 77,78, 79, and 86 for the—in one preferred embodiment combined—treatment ofkidney cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 3, 5, 15, 30, 36, 39, 47, 55, 67, 73, 74, 79, 81, 82, 86, and 87for the—in one preferred embodiment combined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 3, 9, 11, 42, 43, 44, 50, 55, 60, 65, 68, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 81, 82, 83, 84, and 86 for the—in one preferredembodiment combined—treatment of colon cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 3, 9, 11, 42, 43, 44, 50, 55, 60, 65, 68, 70, 71, 72, 73 74, 75,76, 77, 78, 79, 81, 82, 83, 84, and 86 for the—in one preferredembodiment combined—treatment of rectal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 3, 8, 9, 12, 15, 41, 42, 43, 51, 65, 69, 70, 71, 72 73, 74, 75,77, 81, 83, 84, and 85 for the—in one preferred embodimentcombined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 4, 5, 15, 44, 66, 72, 78, and 86 for the—in one preferredembodiment combined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 5, 15, 24, 30, 41, 55, 65, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 83, and 84 for the—in one preferred embodiment combined—treatment ofovarian cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 9, 10, 11, 12, 41, 43, 60, 71, 72, 73, 78, 83, and 84 for the—inone preferred embodiment combined—treatment of breast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 5 30, 44, 55, 58, 65, 67, 68, 69, 71, 72, 73, 74, 76, 79, 81, 82,85, and 86 for the—in one preferred embodiment combined—treatment ofliver cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 10, 15, 24, 30, 34, 44, 45, 50, 68, 70, 71, 72, 73, 74, 78, 81,84, and 86 for the—in one preferred embodiment combined—treatment ofstomach cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 23, 39, 64, 69, 73, 78, 81, and 82 for the—in one preferredembodiment combined—treatment of prostate cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention selected from SEQID No. 16, 30, 66, and 74 for the—in one preferred embodimentcombined—treatment of leukocytic cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length—variant-MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 67.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 67, preferably containing SEQ IDNo. 1 to SEQ ID No. 34, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is for a cellular therapy, a vaccine, aprotein or based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are pancreatic cancer, lung cancer,kidney cancer, brain cancer, colon or rectal cancer, esophageal cancer,breast cancer, ovarian cancer, stomach cancer, liver cancer, prostatecancer, melanoma and leukemias, and preferably pancreatic cancer cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably pancreaticcancer. The marker can be over-presentation of the peptide (s)themselves, or over-expression of the corresponding gene (s). Themarkers may also be used to predict the probability of success of atreatment, preferably an immunotherapy, and most preferred animmunotherapy targeting the same target that is identified by thebiomarker. For example, an antibody or soluble TCR can be used to stainsections of the tumor to detect the presence of a peptide of interest incomplex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Both therapeutic and diagnostic uses against additional cancerousdiseases are disclosed in the following more detailed description of theunderlying expression products (polypeptides) of the peptides accordingto the invention.

The gene for ACAT2 encodes acetyl-CoA acetyltransferase 2, a thiolaseinvolved in lipid metabolism. ACAT2 expression is up-regulated inhepatocellular carcinoma (Song et al., 2006). ACAT2 expression isassociated with radioresistance in pancreatic cancer cell lines (Soucheket al., 2014).

The gene for ACTA1 encodes the skeletal muscle alpha actin, a member ofthe actin family of proteins, which are highly conserved proteins thatplay a role in cell motility, structure and integrity. ACTA1, aclassical myoepithelial marker, was shown to be highly expressed incancer-associated fibroblasts in urinary bladder cancer, oral squamouscell carcinoma, invasive breast cancer, gastric cancer,cholangiocarcinoma and metastatic liver carcinoma and to contribute toepithelial-mesenchymal transition, tumor stroma formation and fibrosis(Schulte et al., 2012; Franz et al., 2010; Kuroda et al., 2005; Nakayamaet al., 2002; Terada et al., 1996).

The gene for ACTA2 encodes the smooth muscle alpha actin, a member ofthe actin family of proteins, which are highly conserved proteins thatplay a role in cell motility, structure and integrity (RefSeq, 2002).Single nucleotide polymorphisms or copy number variations of ACTA2 havebeen identified in chronic lymphocytic leukemia, brain metastases ofnon-small cell lung cancer and cell lines derived from metastaticmelanoma (Berndt et al., 2013; Lee et al., 2012; Dutton-Regester et al.,2012). Functionally, high expression levels of ACTA2 appear to beassociated with enhanced tumor cell invasion and metastasis formation(Kojima et al., 2014; Lee et al., 2013b; Tatenhorst et al., 2004).

The gene for ACTB encodes beta actin, a major constituent of thecontractile apparatus and one of the two non-muscle cytoskeletal actins(RefSeq, 2002). ACTB was shown to be de-regulated in liver cancer,melanoma, renal cancer, colorectal cancer, gastric cancer, pancreaticcancer, esophageal cancer, lung cancer, breast cancer, prostate cancer,ovarian cancer, leukemia and lymphoma. The abnormal expression andpolymerization of ACTB and the resulting changes to the cytoskeletonappear to be associated with the invasiveness and metastasis of cancers(Guo et al., 2013).

The gene for ACTBL2 encodes kappa actin, a member of the actin family ofproteins, which are highly conserved proteins that play a role in cellmotility, structure and integrity (RefSeq, 2002). Increased expressionof ACTBL2 was observed in hepatocellular carcinoma and hepatoma cells,where it altered cell growth properties and contributed to poorpostoperative prognosis (Chang et al., 2006; Chang et al., 2011).

The gene for ACTC1 encodes the cardiac muscle alpha actin 1, which is amajor constituent of the contractile apparatus in cardiac myocytes(RefSeq, 2002). Altered expression of ACTC1 was reported in bladdercancer, paclitaxel-treated non-small lung cancer cells andchemoresistant ovarian cancer (Zaravinos et al., 2011; Che et al., 2013;Pan et al., 2009). Furthermore, ACTC1 might be a useful diagnosticmarker for prostate cancer and rhabdomyosarcoma (Huang et al., 2010;Clement et al., 2003).

The gene for ACTG1 encodes actin gamma 1, a cytoplasmic actin found innon-muscle cells, which acts as mediator of internal cell motility(RefSeq, 2002). ACTG1 was shown to be over-expressed in small cell lungcancer and osteosarcoma and down-regulated in epithelial ovarian cancer(Li et al., 2010; Jeong et al., 2011; Chow et al., 2010). Alterations inACTG1 levels have been reported to promote invasion and metastasisformation in different types of cancer cells. In colon cancer cells andhepatocellular carcinoma cells over-expression of ACTG1 enhancesmigration and invasion, whereas in melanoma cells and salivary glandadenocarcinoma cells down-regulation of ACTG1 is associated with thisphenotype (Simiczyjew et al., 2014; Luo et al., 2014; Zhang et al.,2006; Gutgemann et al., 2001; Suzuki et al., 1998).

The gene for ACTG2 encodes actin gamma 2; a smooth muscle actin found inenteric tissues, which mediates internal cell motility (RefSeq, 2002).ACTG2 is discussed as potential biomarker for prostate cancer diagnosisand was shown to be up-regulated in transdifferentiated prostate stromalcells (Fillmore et al., 2014; Untergasser et al., 2005). Regardingchemotherapy, ACTG2 is up-regulated upon paclitaxel treatment oflaryngeal cancer cells, appears to be implicated in cisplatin resistancein breast cancer cells and was shown to positively correlate with thesensitivity of colorectal cancer with liver metastases to the FOLFOX4regimen (Xu et al., 2013; Watson et al., 2007; Lu et al., 2013b).

The gene for ADAM8 encodes ADAM metallopeptidase domain 8, a member ofthe disintegrin and metalloprotease domain family that is involved incell-cell and cell-matrix interactions (RefSeq, 2002). ADAM8over-expression in pancreatic cancer is associated with increasedmigration and invasiveness of pancreatic ductal adenocarcinoma cells(Schlomann et al., 2015). ADAM8 is involved in tumor cell migration andinvasion in lung cancer, renal cell carcinoma and brain cancers(Mochizuki and Okada, 2007).

The gene for AEBP1 encodes adipocyte enhancer binding protein 1, acarboxypeptidase A that may function as a transcriptional co-repressorwith importance for adipogenesis and smooth muscle cell differentiation(RefSeq, 2002). AEBP1 is up-regulated in melanoma and contributes toacquired resistance to mutant v-raf murine sarcoma viral oncogenehomolog B1 (BRAF) inhibition (Hu et al., 2013). AEBP1 is up-regulated inthe majority of primary glioblastoma (Reddy et al., 2008).

The gene for AHNAK2 encodes the scaffold protein AHNAK nucleoprotein 2(Marg et al., 2010). AHNAK2 is an important element of the non-classicalsecretion pathway of fibroblast growth factor 1 (FGF1), a factorinvolved in tumor growth and invasion (Kirov et al., 2015).

The gene for ANKH encodes ankylosis, progressive homolog (mouse)/ANKHinorganic pyrophosphate transport regulator, a multipass transmembraneprotein that controls pyrophosphate levels (RefSeq, 2002).

The gene for ANO1 encodes anoctam in 1, a calcium-activated chloridechannel associated with small intestinal sarcoma and oral cancer(RefSeq, 2002). ANO1 is amplified in esophageal squamous cell cancer(ESCC), gastrointestinal stromal tumor (GIST), head and neck squamouscell carcinoma (HNSCC), pancreatic and breast cancers (Qu et al., 2014).

The gene for APOB encodes apolipoprotein B, the main apolipoprotein ofchylomicrons and low density lipoproteins (LDH) (RefSeq, 2002). Inalpha-fetoprotein-negative HBV-related HCC, APOB was found to be one ofthe 14 differentially expressed proteins which could be associated withHCC progression (He et al., 2014). In advanced breast cancer, APOB wasfound to be the one of 6 differentially expressed proteins which couldpredict the responsiveness to neoadjuvant chemotherapy and relapse-freesurvival of patients (Hyung et al., 2011).

The gene for ASPHD1 encodes aspartate beta-hydroxylase domaincontaining 1. ASPHD1 is located on chromosome 16p11.2 (RefSeq, 2002).

The gene for ATM encodes ataxia telangiectasia mutated, a PI3/PI4-kinasefamily member and master controller of cell cycle checkpoint signalingpathways that are required for cell response to DNA damage and forgenome stability (RefSeq, 2002). ATM is a tumor suppressor which isfrequently mutated in a broad range of human cancers including lung,colorectal, breast and hematopoietic cancers (Weber and Ryan, 2014).

The gene for ATP5B encodes ATP synthase, H+ transporting, mitochondrialF1 complex, beta polypeptide, the beta subunit of the catalytic core ofmitochondrial ATP synthase (RefSeq, 2002). ATP5B gene expression wassignificantly higher in colorectal cancer tissues compared to healthytissues (Geyik et al., 2014). ATP5B down-regulation in tumor tissues isclosely related to the metastasis, invasion, and poor-prognosis ofgallbladder cancer (Sun et al., 2015b).

The gene for ATP5L encodes ATP synthase, H+ transporting, mitochondrialFo complex, subunit G of the membrane-spanning component of themitochondrial ATP synthase, which comprises the proton channel (RefSeq,2002).

The gene for ATP5L2 encodes ATP synthase, H+ transporting, mitochondrialFo complex, subunit G2 of the membrane-spanning component of themitochondrial ATP synthase, which comprises the proton channel (RefSeq,2002).

The gene for BACE2 encodes beta-site APP-cleaving enzyme 2, an integralmembrane glycoprotein and aspartic protease. BACE2 cleaves amyloidprecursor protein into amyloid beta peptide (RefSeq, 2002). BACE2 isinvolved in pancreatic beta-cell function (Vassar et al., 2014).

The gene for CCNB1 encodes cyclin B1, a regulatory protein involved inmitosis (RefSeq, 2002). CCNB1 is a well-described tumor antigen andCCNB1 over-expression has been described for breast, head and neck,prostate, colorectal, lung and liver cancers (Egloff et al., 2006).

The gene for CEACAM6 encodes carcinoembryonic antigen-related celladhesion molecule 6 (non-specific cross reacting antigen), a member ofthe CEACAM family of tumor markers (RefSeq, 2002). CEACAM6 isup-regulated in gastric cancers (Yasui et al., 2004). CEACAM6 is acandidate breast tumor antigen (Sood, 2010).

The gene for CLTA encodes clathrin, light chain A, a structuralcomponent of coated pits with regulatory function (RefSeq, 2002). TheCLTA gene shows an alternative splice pattern in glioma (Cheung et al.,2008).

The gene for CTLB encodes clathrin, light chain B, a structuralcomponent of coated pits with regulatory function (RefSeq, 2002).

The gene for CLU encodes a secreted chaperone that might be involved inseveral basic biological events such as cell death, tumor progression,and neurodegenerative disorders (RefSeq, 2002). Its role intumorigenesis appears to be ambivalent as in normal cells and duringearly phases of carcinogenesis, CLU may inhibit tumor progression,whereas in advanced neoplasia, it may offer a significant survivaladvantage in the tumor by suppressing many therapeutic stressors andenhancing metastasis. CLU has been shown to play a critical role inprostate cancer pathogenesis, to regulate the aggressive behavior ofhuman clear renal cell carcinoma cells through modulating ERK1/2signaling and MMP-9 expression and to confer resistance to treatment inadvanced stages of lung cancer (Trougakos, 2013; Panico et al., 2009;Takeuchi et al., 2014; Wang et al., 2014b).

The gene for COL12A1 encodes the alpha chain of type XII collagen, amember of the FACIT (fibril-associated collagens with interrupted triplehelices) collagen family and thus is a part of extracellular matrix(ECM) (RefSeq, 2002). COL12A1 is over-expressed in drug-resistantvariants of ovarian cancer cell lines (Januchowski et al., 2014). Incolorectal cancer, COL12A1 is over-expressed in desmoplastic stroma byand around cancer-associated fibroblasts, as well as in cancer cellslining the invasion front (Karagiannis et al., 2012).

The gene for COL6A3 encodes the alpha-3 chain of type VI collagen, abeaded filament collagen found in most connective tissues, playing animportant role in the organization of matrix components (RefSeq, 2002).COL6A3 expression was reported to be increased in pancreatic cancer,colon cancer, gastric cancer, mucoepidermoid carcinomas and ovariancancer. Cancer associated transcript variants including exons 3, 4 and 6were detected in colon cancer, bladder cancer, prostate cancer andpancreatic cancer (Arafat et al., 2011; Smith et al., 2009; Yang et al.,2007; Xie et al., 2014; Leivo et al., 2005; Sherman-Baust et al., 2003;Gardina et al., 2006; Thorsen et al., 2008). In ovarian cancer COL6A3levels correlated with higher tumor grade and in pancreatic cancerCOL6A3 was shown to represent a suitable diagnostic serum biomarker(Sherman-Baust et al., 2003; Kang et al., 2014).

The gene for DCBLD2 encodes discoidin, CUB and LCCL domain-containingprotein 2 also referred to as endothelial and smooth muscle cell-derivedneuropilin-like protein, a transmembrane co-receptor protein (RefSeq,2002). DCBLD2 is up-regulated in glioblastomas and head and neck cancers(HNCs) and is required for EGFR-stimulated tumorigenesis (Feng et al.,2014). Furthermore, DCBLD2 is up-regulated in highly metastatic lungcancer sublines and tissue samples (Koshikawa et al., 2002). Incontrast, the expression of DCBLD2 is silenced by hypermethylation ofits promoter in gastric cancer (Kim et al., 2008).

The gene for DUSP14, the dual-specificity phosphatase 14, cande-phosphorylate tyrosine as well as serine/threonine residues and playsa role in the inactivation of MAP kinase signaling (RefSeq, 2002).Single nucleotide polymorphisms in the DUSP14 gene are associated withaltered melanoma risk (Yang et al., 2014a; Liu et al., 2013a).

The gene for EEF1A1 encodes an isoform of the alpha subunit of theelongation factor-1 complex, which is responsible for the enzymaticdelivery of aminoacyl tRNAs to the ribosome (RefSeq, 2002). EEF1A1 wasshown to be up-regulated in a variety of cancer entities, includingcolorectal cancer, ovarian cancer, gastric cancer, prostate cancer,glioblastoma and squamous cell carcinoma and was described as potentialserum biomarker for prostate cancer (Matassa et al., 2013; Vui-Kee etal., 2012; Lim et al., 2011; Kuramitsu et al., 2010; Kido et al., 2010;Scrideli et al., 2008; Qi et al., 2005; Rehman et al., 2012).Mechanistically, EEF1A1 inhibits apoptosis through an interaction withp53 and p73, promotes proliferation by transcriptional repression of thecell cycle inhibitor p21 and participates in the regulation ofepithelial-mesenchymal transition (Blanch et al., 2013; Choi et al.,2009; Hussey et al., 2011).

The gene for EEF1A1P5 encodes eukaryotic translation elongation factor 1alpha 1 pseudogene 5 and is located on chromosome 9q34.13 (RefSeq,2002).

The gene for FAMC3 is a member of the family with sequence similarity 3(FAM3) family and encodes a secreted protein with a GG domain. A changein expression of this protein has been noted in pancreaticcancer-derived cells (RefSeq, 2002). In melanoma, FAMC3 has beenidentified as a candidate biomarker for autophagy, an important tumorcell survival mechanism (Zou et al., 2002; Kraya et al., 2015). FAMC3plays an essential role in the epithelial-mesenchymal transition whichcorrelates with aggressiveness, metastatic progression of tumors andpoor survival especially in hepatocellular cancer, colorectal cancer,lung and breast cancers (Csiszar et al., 2014; Gao et al., 2014c; Songet al., 2014; Chaudhury et al., 2010; Lahsnig et al., 2009).

The gene for FAP encodes a transmembrane serine protease which isselectively expressed in reactive stromal fibroblasts of epithelialcancers (cancer-associated fibroblasts or CAFs), granulation tissue ofhealing wounds, and malignant cells of bone and soft tissue sarcomas(RefSeq, 2002). FAP plays an important role in cancer growth andmetastasis through its involvement in cell adhesion, migration processesand remodeling of the extracellular matrix (ECM) (Jacob et al., 2012).The over-expression of FAP correlates with poor prognosis, advancedtumor staging, metastasis formation and invasive potential in variouscancers, thereunder in colon cancer, esophageas squamous cell carcinoma,pancreatic adenocarcinoma, glioblastoma, osteosarcoma, ovarian cancerand breast cancer (Wikberg et al., 2013; Kashyap et al., 2009; Cohen etal., 2008; Mentlein et al., 2011; Yuan et al., 2013; Zhang et al., 2011;Ariga et al., 2001).

The gene for FKBP10 encodes the FK506 binding protein 10, which belongsto the FKBP-type peptidyl-prolyl cis/trans isomerase family. The FKBP10gene product localizes to the endoplasmic reticulum and acts as amolecular chaperone (RefSeq, 2002). FKBP10 was identified as a novelgene that participates in the acquisition and maintenance of theadriamycin-resistant phenotype in leukemia cells (Sun et al., 2014).FKBP10 has been associated with colorectal cancer through itsup-regulation (Olesen et al., 2005). In contrast, the under-expressionof FKBP10 was characteristic for epithelial ovarian carcinomas (Quinn etal., 2013).

The gene for FLNA encodes filamin A, an actin-binding protein thatcrosslinks actin filaments and links actin filaments to membraneglycoproteins. The encoded protein is involved in the remodeling of thecytoskeleton which induces changes in cell shape and migration andinteracts with integrins, transmembrane receptor complexes, and secondmessengers (RefSeq, 2002). Depending on its subcellular localization,filamin A plays a dual role in cancer: In the cytoplasm, filamin Afunctions in various growth signaling pathways, in addition to beinginvolved in cell migration and adhesion pathways. Thus, itsover-expression has a tumor-promoting effect. In contrast to full-lengthfilamin A, the C-terminal fragment, which is released upon proteolysisof the protein, localizes to the nucleus, where it interacts withtranscription factors and thereby suppresses tumor growth and metastasis(Savoy and Ghosh, 2013).

The gene for GGA1 encodes a member of the Golgi-localized, gamma adaptinear-containing, ARF-binding (GGA) protein family. Members of this familyare ubiquitous coat proteins that regulate the trafficking of proteinsbetween the trans-Golgi network and the lysosome (RefSeq, 2002).

The gene for HBB encodes the beta chain of human hemoglobin, theiron-containing oxygen-transport metalloprotein in the red blood cell(RefSeq, 2002). The ability of breast cancer to generate bone andvisceral metastases represents a clear indication of poor clinicaloutcome compared to cases of breast cancer with metastasis restricted tobone. The increased expression of HBB in bone metastasis correlated withtheir ability to rapidly spread to other organs (Capulli et al., 2012).HBB was shown to be over-expressed in uterine cervix carcinoma tissue.The ectopic expression of HBB in cervical cancer cells suppressedoxidative stress and improved cell viability (Li et al., 2013).

The gene for HBD encodes the delta chain of human hemoglobin, theiron-containing oxygen-transport metalloprotein in the red blood cell.Two alpha chains plus two delta chains constitute hemoglobin A2, whichwith HbF comprises 3% of adult hemoglobin (RefSeq, 2002).

The gene for HKDC1 encodes hexokinase domain containing 1, whichexhibits the hexokinase activity in vitro (Guo et al., 2015). Using anovel method to identify potential therapeutic targets fromheterogeneous data, HKDC1, among other well-known therapeutic targets,was discovered as a novel potential therapeutic target for lung cancer(Li and Huang, 2014).

The gene for HSPD1 encodes a mitochondrial heat shock 60 kDa protein 1,a member of the chaperonin family, which is essential for the foldingand assembly of newly imported proteins in the mitochondria and mayfunction as a signaling molecule in the innate immune system (RefSeq,2002). Although HSPD1 is considered an intramitochondrial protein, ithas been found in the cytosol, cell membrane, vesicles, cell surface,extracellular space, and blood. As cytosolic HSPD1 levels graduallyincrease or decrease during carcinogenesis in various organs, HSPD1 canbe used as a biomarker for the diagnosis and prognosis of pre-neoplasticand neoplastic lesions. Furthermore, some newly identified functions ofHSPD1 are associated with carcinogenesis, specifically with tumor cellsurvival and proliferation and it has been intensively discussed as apromising target for anti-tumor therapy (Pace et al., 2013; Nakamura andMinegishi, 2013; Cappello et al., 2013; Cappello et al., 2011; Cappelloet al., 2008).

The gene for HYOU1 encodes hypoxia up-regulated 1 protein, better knownas 170 kDa glucose-regulated protein (GRP170), which belongs to the heatshock protein 70 family. The expression of HYOU1 is induced instress-dependent manner under hypoxic conditions and results in theaccumulation of the protein in the endoplasmic reticulum (ER). Theprotein encoded by HYOU1 is thought to play an important role in proteinfolding and secretion in the ER (RefSeq, 2002). The activity ofintracellular HYOU1 protein has been shown to provide a survival benefitin cancer cells during tumor progression or metastasis. Theextracellular HYOU1 protein plays an essential role in the generation ofan anti-tumor immune response by facilitating the delivery of tumorantigens for their cross-presentation (Fu and Lee, 2006; Wang et al.,2014a). HYOU1 protein has been introduced in cancer immunotherapy andshowed a positive immunomodulating effect (Yu et al., 2013; Chen et al.,2013a; Yuan et al., 2012; Wang and Subjeck, 2013). In prostate cancercells, the suppression of HYOU1 showed an anti-tumor effect (Miyagi etal., 2001).

The gene for IFT88 encodes a member of the tetratrico peptide repeat(TPR) family (RefSeq, 2002). In mitosis, IFT88 is part of adynein1-driven complex that transports peripheral microtubule clustersto spindle poles to ensure proper spindle orientation. IFT88 depletioninduces mitotic defects in human cultured cells (Delaval et al., 2011).Loss of IFT88 (also called Tg737) gene expression results in theproliferation of liver stem cells (oval cells) and is therefore a liverneoplasia tumor suppressor gene (Isfort et al., 1997). In 2012 amutation was found to be responsible for a novel form of ciliopathy andanosmia in humans capable of remedy in mice by adenoviral mediated genetherapy (McIntyre et al., 2012).

The gene for IGF2BP3 encodes insulin-like growth factor II mRNA bindingprotein 3, an oncofetal protein, which represses translation ofinsulin-like growth factor II (RefSeq, 2002). Several studies have shownthat IGF2BP3 acts in various important aspects of cell function, such ascell polarization, migration, morphology, metabolism, proliferation anddifferentiation. In vitro studies have shown that IGF2BP3 promotes tumorcell proliferation, adhesion, and invasion. Furthermore, IGF2BP3 hasbeen shown to be associated with aggressive and advanced cancers (Bellet al., 2013; Gong et al., 2014). IGF2BP3 over-expression has beendescribed in numerous tumor types and correlated with poor prognosis,advanced tumor stage and metastasis, as for example in neuroblastoma,colorectal carcinoma, intrahepatic cholangiocarcinoma, hepatocellularcarcinoma, prostate cancer, and renal cell carcinoma (Bell et al., 2013;Findeis-Hosey and Xu, 2012; Hu et al., 2014; Szarvas et al., 2014; Jenget al., 2009; Chen et al., 2011; Chen et al., 2013b; Hoffmann et al.,2008; Lin et al., 2013b; Yuan et al., 2009).

The gene for ITGB4 encodes for a protein of the Integrin family.Integrins are heterodimers comprised of alpha and beta subunits that arenon-covalently associated transmembrane glycoprotein receptors. Theymediate cell-matrix or cell-cell adhesion, and transduce signals thatregulate gene expression and cell growth (RefSeq, 2002). ITGB4 (alsocalled CD104) tends to associate with the alpha 6 subunit and is likelyto play a pivotal role in the biology of several invasive carcinomassuch as esophageal squamous cell carcinoma, bladder and ovariancarcinoma (Kwon et al., 2013; Pereira et al., 2014; Chen et al., 2014b).A single nucleotide polymorphism in ITGB4 seems to influence tumoraggressiveness and survival and may have prognostic value for breastcancer patients (Brendle et al., 2008).

The gene for KCNK6 encodes one of the members of the superfamily ofpotassium channel proteins containing two pore-forming P domains. Thischannel protein, considered an open rectifier, is widely expressed. Itis stimulated by arachidonic acid, and inhibited by internalacidification and volatile anesthetics (RefSeq, 2002). KCNK6 (alsocalled K2P6.1) together with K2P1.1, K2P3.1, K2P5.1, K2P6.1, K2P7.1 andK2P10.1 showed significant under-expression across the cancer typesexamined using the online cancer microarray database, Oncomine(www.oncomine.org) (Williams et al., 2013).

The gene for KCNN3 belongs to the KCNN family of potassium channels. Itencodes an integral membrane protein that forms a voltage-independentcalcium-activated channel, which is thought to regulate neuronalexcitability by contributing to the slow component of synaptic afterhyperpolarization (RefSeq, 2002). KCNN3 (also called TASK-1) expressionwas down-regulated by 17beta-estradiol in mouse neuroblastoma N2A cellsand improved cell proliferation (Hao et al., 2014). KCNN3 expression wasup-regulated by exposure of breast cancer organotypic culture to 1,25dihydroxy vitamin D (3) in physiological and supra-physiologicalconcentrations (Milani et al., 2013). KCNN3 (also called K2P3.1)together with K2P1.1 and K2P12.1, were over-expressed in a range ofcancers examined using the online cancer microarray database, Oncomine(www.oncomine.org) (Williams et al., 2013).

The gene for KDM1A (also called LSD1) encodes a nuclear proteincontaining a SWIRM domain, a FAD-binding motif, and an amine oxidasedomain. This protein is a component of several histone deacetylasecomplexes, though it silences genes by functioning as a histonedemethylase (RefSeq, 2002). Over-expression of KDM1A promotes tumor cellproliferation, migration and invasion and was associated with poorprognosis in NSCLC and HCC (Lv et al., 2012; Zhao et al., 2013).Elevated expression of KDM1A correlates with prostate cancer recurrenceand with increased VEGF-A expression (Kashyap et al., 2013). Inhibitionof KDM1A with a combination of trichostatin A (TSA) and5-aza-2′-deoxycytidine (decitabine) suppresses the tumorigenicity of theovarian cancer ascites cell line SKOV3 (Meng et al., 2013).

The gene for KIF26B encodes for a member of the kinesin superfamilyproteins (KIFs) which is essential for kidney development. KIF26Bexpression is restricted to the metanephric mesenchyme, and itstranscription is regulated by a zinc finger transcriptional regulatorSaII1 (Terabayashi et al., 2012). High expression of KIF26B in breastcancer associates with poor prognosis (Wang et al., 2013b). KIF26Bup-regulation was significantly correlated with tumor size analyzing CRCtumor tissues and paired adjacent normal mucosa. KIF26B plays animportant role in colorectal carcinogenesis and functions as a novelprognostic indicator and a potential therapeutic target for CRC (Wang etal., 2015).

The gene for KRT19 encodes a member of the keratin family. The keratinsare intermediate filament proteins responsible for the structuralintegrity of epithelial cells and are subdivided into cytokeratins andhair keratins. KRT19 is specifically expressed in the periderm, thetransiently superficial layer that envelopes the developing epidermis(RefSeq, 2002). KRT19 expression in tumor cells is a prognostic markerfor several tumor entities such as breast, lung, ovarian andhepatocellular cancer (Skondra et al., 2014; Gao et al., 2014b; Liu etal., 2013b; Lee et al., 2013a). KRT19 has been shown to be anindependent prognostic factor for pancreatic neuroendocrine tumors,especially the insulin-negative tumors. KRT19 positive tumors areassociated with poor outcome irrespective of the established pathologicparameters such as size, mitoses, lymphovascular invasion, and necrosis(Jain et al., 2010).

The gene for KRT7 encodes a member of the keratin gene family. The typeII cytokeratins consist of basic or neutral proteins which are arrangedin pairs of heterotypic keratin chains co-expressed duringdifferentiation of simple and stratified epithelial tissues. This typeII cytokeratin is specifically expressed in the simple epithelia liningthe cavities of the internal organs and in the gland ducts and bloodvessels (RefSeq, 2002). KRT7 is used in immunohistochemistry todifferentiate between several phenotypes and as biomarker for prognosisof certain cancers as renal cell carcinoma, ovarian carcinoma,epithelial skin tumor etc. (Kuroda et al., 2013; McCluggage and Young,2005; Alhumaidi, 2012).

The gene for LAMC2 belongs to the family of laminins, a family ofextracellular matrix glycoproteins. Laminins are the majornon-collagenous constituent of basement membranes. They have beenimplicated in a wide variety of biological processes including celladhesion, differentiation, migration, signaling, neurite outgrowth andmetastasis. LAMC2 encodes a protein which is expressed in several fetaltissues and is specifically localized to epithelial cells in skin, lungand kidney (RefSeq, 2002). LAMC2 is highly expressed in anaplasticthyroid carcinoma and is associated with tumor progression, migration,and invasion by modulating signaling of EGFR (Garg et al., 2014). LAMC2expression predicted poorer prognosis in stage II colorectal cancerpatients (Kevans et al., 2011). LAMC2 expression together with threeother biomarkers was found to be significantly associated with thepresence of LN metastasis in oral squamous cell carcinoma patients(Zanaruddin et al., 2013).

The gene for LUM encodes a member of the small leucine-rich proteoglycan(SLRP) family that includes decorin, biglycan, fibromodulin, keratocan,epiphycan, and osteoglycin. Lumican is the major keratan sulfateproteoglycan of the cornea but is also distributed in interstitialcollagenous matrices throughout the body. Lumican may regulate collagenfibril organization and circumferential growth, corneal transparency,and epithelial cell migration and tissue repair (RefSeq, 2002). LUMprotein is up-regulated in most tumor tissues such as breast cancer,colorectal cancer and pancreatic cancer compared to normal tissue and isassociated with higher tumor grade and poor outcome. Howeverextracellular lumican inhibits pancreatic cancer cell growth and isassociated with prolonged survival after surgery (Leygue et al., 1998;Seya et al., 2006; Ishiwata et al., 2007; Li et al., 2014). LUM andother genes related to extracellular matrix integrity (DCN and DPT) aredifferentially expressed and may serve as biomarkers for metastatic andrecurrent giant cell tumor of bone (Lieveld et al., 2014). LUM isdown-regulated in cisplatin-, doxorubicin-, topotecan-, andpaclitaxel-resistant variants of the A2780 ovarian cancer cell line(Januchowski et al., 2014).

The gene for MAP4 encodes a major non-neuronal microtubule-associatedprotein, which promotes microtubule assembly and counteractsdestabilization of interphase microtubule catastrophe promotion.Phosphorylation of this protein affects microtubule properties and cellcycle progression (RefSeq, 2002). High levels of MAP4 were shown topositively correlate with bladder cancer grade, whereas phosphorylationof the protein by protein kinase A reduces bladder cancer cell migrationand invasion (Ou et al., 2014). A study in non-small cell lung cancerpatients reported an increased ratio of MAP4 to stathmin mRNA in tumorsamples compared to normal samples, indicating that this ratio couldserve as biomarker for non-small cell lung cancer (Cucchiarelli et al.,2008). MAP4 levels, which are negatively regulated by the tumorsuppressor p53, influence the efficacy of microtubule-targeting agents.High levels increase the effect of microtubule stabilizing drugs(taxanes) and reduce the effect of microtubule destabilizing drugs(vinca alcaloids), while low MAP4 levels have the opposite effect (Haitand Yang, 2006; Galmarini et al., 2003; Zhang et al., 1999).

The gene for MMP7 encodes an enzyme that degrades proteoglycans,fibronectin, elastin and casein and differs from most MMP family membersin that it lacks a conserved C-terminal protein domain. Proteins of thematrix metalloproteinase (MMP) family are involved in the breakdown ofextracellular matrix in normal physiological processes, such asembryonic development, reproduction, and tissue remodeling, as well asin disease processes, such as arthritis and metastasis (RefSeq, 2002).MMP7 is frequently over-expressed in human cancer tissue, includingcolorectal cancer, metastatic lung carcinoma and gastric cancer and isassociated with cancer progression and metastasis formation (Ii et al.,2006; Sun et al., 2015a; Han et al., 2015; Long et al., 2014). MMP7 hasbeen shown to play important tumor promoting roles, like degradation ofextracellular matrix proteins, activation of tumor cell proliferation byincreasing the bioavailability of insulin-like growth factor andheparin-binding epidermal growth factor and induction of apoptosis intumor-adjacent cells by cleaving membrane bound Fas ligand (Ii et al.,2006).

The gene for MROH6, also known as C8orf73, is located on chromosome8q24.3 (RefSeq, 2002).

The gene for MX1 encodes a guanosine triphosphate (GTP)-metabolizingprotein that is induced by type I and type II interferons andparticipates in the cellular antiviral response (RefSeq, 2002). The roleof MX1 in cancer is not fully elucidated yet. On the one hand MX1expression inversely correlates with prostate cancer, reduces metastasisformation and enhances the sensitivity to docletaxel. Furthermore,epigenetic silencing of MX1 by hypermethylation has been detected inhead and neck squamous cell carcinoma and MX1 expression reduces cellmotility and invasion of prostate cancer and melanoma cell lines, allfavoring tumor suppressive actions of MX1 (Brown et al., 2015; Calmon etal., 2009; Mushinski et al., 2009). On the other hand, a singlenucleotide polymorphism within the MX1 gene is associated with prostatecancer and high expression of MX1 is associated with lymph nodemetastasis in colorectal cancer, which indicates oncogenic properties ofMX1 (Croner et al., 2014; Glymph et al., 2013).

The gene for MXRA5 encodes one of the matrix-remodeling associatedproteins, which contains 7 leucine-rich repeats and 12immunoglobulin-like C2-type domains related to perlecan (RefSeq, 2002).A Chinese study identified MXRA5 as the second most frequently mutatedgene in non-small cell lung cancer (Xiong et al., 2012). In coloncancer, MXRA5 was shown to be over-expressed and might serve as abiomarker for early diagnosis and omental metastasis (Zou et al., 2002;Wang et al., 2013a).

The gene for MYH9 encodes a conventional non-muscle myosin IIA heavychain that contains an IQ domain and a myosin head-like domain which isinvolved in several important functions, including cytokinesis, cellmotility and maintenance of cell shape (RefSeq, 2002). High expressionof MYH9 was shown to be associated with poor prognosis in esophagealsquamous cell carcinoma and, in combination with annexin II andkindling-2, might serve as predictive biomarker for overall and diseasefree survival in this disease (Xia et al., 2012; Cao et al., 2014).Mutations within the MYH9 gene have been identified in human breastcancer samples and it is differentially expressed in colon carcinoma(Ellis et al., 2012; Mu et al., 2013). In vitro and xenograft studiesindicate that MYH9 promotes tumor cell growth and invasion of differenttumor cell lines, including breast cancer and non-small lung cancercells (Robinson et al., 2013; Lin et al., 2013a; Lund et al., 2012;Derycke et al., 2011; Medjkane et al., 2009).

The gene for MYL12A encodes a non-sarcomeric myosin regulatory lightchain, which regulates smooth muscle and non-muscle cell contraction(Amatschek et al., 2004; RefSeq, 2002). Phosphorylation of MYL12A wasreported to promote tumor cell motility and invasion in vitro and in theanimal model (Manning, Jr. et al., 2000; Kaneko et al., 2002; Khuon etal., 2010). Furthermore, MYL12A appears to regulate DNA damage repairand p53-driven apoptosis, by sequestering the transcriptional regulatorapoptosis-antagonizing transcription factor (Hopker et al., 2012a;Hopker et al., 2012b).

The gene for MYL12B encodes a regulatory light chain of the non-musclemyosin II (MYH9). Phosphorylation of MYL12B results in higher MgATPaseactivity and the assembly of myosin II filaments (RefSeq, 2002). Theprotein was shown to be up-regulated in grade 3 ovarian cancer andpharmacologic block of MYL12B phosphorylation or activation decreasedtumor cell migration and invasion in vitro and metastasis formation inan animal model for breast cancer. These data indicate a pro-metastaticrole for MYL12B (Lim et al., 2011; Menhofer et al., 2014; Zhang et al.,2013; Patel et al., 2012).

The gene for PARD3B encodes a protein that localizes to tight junctionsof epithelial cells and participates in the establishment of cellpolarity (Izaki et al., 2005). A single nucleotide polymorphism withinthe PARD3B gene was shown to be significantly associated with severetreatment-related hepatotoxicity in children with acute lymphoblasticleukemia or lymphoblastic lymphoma (Horinouchi et al., 2010).

The gene for PDIA6 (also called ERp5) encodes a protein disulfideisomerase which is an endoplasmic reticulum (ER) resident protein thatcatalyzes formation, reduction, and isomerization of disulfide bonds inproteins and is thought to play a role in folding of disulfide-bondedproteins (RefSeq, 2002). Immunostaining of prostate tissue microarraysfor PDIA6 showed a significantly higher immunoreactivity inpre-malignant lesions compared with non-malignant epithelium (P<0.0001,Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade(2-3) cancers (P<0.05) (Glen et al., 2010). High ERp5/ADAM10 expressionleads to MICA shedding and impaired NKG2D ligands recognition in lymphnode microenvironment in Hodgkin lymphomas. This leads todown-modulation of NKG2D surface expression on CD8 T cells and aninefficient anti-tumor response (Zocchi et al., 2012). The proteindisulfide isomerases PDIA4 and PDIA6 mediate resistance tocisplatin-induced cell death in lung adenocarcinoma (Horibe et al.,2014).

The gene for PIK3IP1 encodes phosphoinositide-3-kinase interactingprotein 1, a PI3K inhibitor (RefSeq, 2002). PIK3IP1 down-regulationleads to increased tumor growth in human T-cell lymphoblastic lymphomacells (Wong et al., 2014). PIK3IP1 is down-regulated in hepatocellularcarcinoma (HCC) and PIK3IP1 suppresses the development of HCC (He etal., 2008).

The gene for PLEC encodes the plakin family member plectin, a proteininvolved in the cross-linking and organization of the cytoskeleton andadhesion complexes (Bouameur et al., 2014). PLEC is over-expressed incolorectal adenocarcinoma, head and neck squamous cell carcinoma andpancreatic cancer (Lee et al., 2004; Katada et al., 2012; Bausch et al.,2011).

The gene for POTEE encodes POTE ankyrin domain family, member E, one of13 paralogs belonging to the POTE gene family. POTE genes are thought torepresent a new family of cancer-testis antigens. The biologicalfunction of the POTE gene family is not fully elucidated yet, but someevidence suggests a pro-apoptotic role (Liu et al., 2009; Bera et al.,2006). POTEE is predominantly expressed in prostate, breast, colon, lungand ovarian cancer (Bera et al., 2006). One study described POTEE to beclosely related to breast cancer, using a combined transcriptomic andproteomic approach (Cine et al., 2014).

The gene for POTEF encodes POTE ankyrin domain family, member J, one of13 paralogs belonging to the POTE gene family. POTE genes are thought torepresent a new family of cancer-testis antigens. The biologicalfunction of the POTE gene family is not fully elucidated yet, but someevidence suggests a pro-apoptotic role (Liu et al., 2009; Bera et al.,2006). POTEF was shown to induce apoptosis in Hela cells through amitochondrial pathway (Liu et al., 2009). POTEF is predominantlyexpressed in prostate, breast, colon, lung and ovarian cancer (Bera etal., 2006).

The gene for POTEI is located on chromosome 2q21.1 and encodes POTEankyrin domain family, member I, one of 13 paralogs belonging to thePOTE gene family. POTE genes are thought to represent a new family ofcancer-testis antigens. The biological function of the POTE gene familyis not fully elucidated yet, but some evidence suggests a pro-apoptoticrole (Liu et al., 2009; Bera et al., 2006). POTEI is predominantlyexpressed in prostate, breast, colon, lung and ovarian cancer (Bera etal., 2006).

The gene for POTEJ encodes POTE ankyrin domain family, member J, one of13 paralogs belonging to the POTE gene family. POTE genes are thought torepresent a new family of cancer-testis antigens. The biologicalfunction of the POTE gene family is not fully elucidated yet, but someevidence suggests a pro-apoptotic role (Liu et al., 2009; Bera et al.,2006). POTEJ is predominantly expressed in prostate, breast, colon, lungand ovarian cancer (Bera et al., 2006).

The gene for POTEKP encodes POTE ankyrin domain family, member K,pseudogene and is located on chromosome 2q21.1 (RefSeq, 2002).

The gene for POTEM encodes POTE ankyrin domain family, member M, one of13 paralogs belonging to the POTE gene family. POTE genes are thought torepresent a new family of cancer-testis antigens. The biologicalfunction of the POTE gene family is not fully elucidated yet, but someevidence suggests a pro-apoptotic role (Liu et al., 2009; Bera et al.,2006). POTEM was identified as specific transcript for normal andmalignant prostate tissue (Stolk et al., 2004).

The gene for PTRF encodes polymerase I and transcript release factor, aregulator of rRNA transcription that promotes the dissociation oftranscription complexes and the re-initiation of polymerase I on nascentrRNA transcripts (RefSeq, 2002). PTRF is down-regulated in breast cancercell lines and breast tumor tissue (Bai et al., 2012). PTRF is anon-small cell lung cancer biomarker (Gamez-Pozo et al., 2012). PTRFexpression is down-regulated in prostate cancer and the absence of PTRFin prostate cancer cells contributes significantly to tumor progressionand metastasis by promoting the angiogenic potential of cancer cells(Nassar et al., 2013).

The gene for PUS7L encodes pseudouridylate synthase 7 homolog (s.cerevisiae)-like, a protein with possible pseudouridine synthaseactivity. The PUS7L gene is located on chromosome 12q12 (RefSeq, 2002).

The gene for RAN encodes RAN, member RAS oncogene family, a small GTPbinding protein that is involved in the translocation of RNA andproteins through the nuclear pore complex, in the control of DNAsynthesis and cell cycle progression, in the formation and organizationof the microtubule network, and in the activation of the androgenreceptor (RefSeq, 2002). RAN is a key protein in the metastaticprogression of cancer. RAN is over-expressed in a range of tumors, suchas breast and renal (Matchett et al., 2014).

The gene for RANP1 encodes RAN, member RAS oncogene family pseudogene 1,a pseudogene located on chromosome 6p21.33 (RefSeq, 2002).

The gene for RASA4 encodes RAS p21 protein activator 4, a Ca(2+)-dependent Ras GTPase-activating protein that switches off theRas-MAPK pathway in response to Ca (2+) (RefSeq, 2002). RASA4 issignificantly amplified in primary effusion lymphoma (Roy et al., 2011).RASA4 is differentially expressed in endometrial adenocarcinoma comparedto normal endometrium (Jeda et al., 2014).

The gene for RASA4B encodes RAS p21 protein activator 4B, a Ca(2+)-dependent Ras GTPase-activating protein with possible involvementin the regulation of the Ras-MAPK pathway (RefSeq, 2002).

The gene for RCN1 encodes reticulocalbin 1, EF-hand calcium bindingdomain, a calcium-binding protein located in the lumen of theendoplasmic reticulum. RCN1 is localized to the plasma membrane in humanendothelial and prostate cancer cell lines (RefSeq, 2002). RCN1 isover-expressed in breast cancer (Amatschek et al., 2004).

The gene for RGS4 encodes regulator of G-protein signaling 4, a GTPaseactivating protein (GAP) for G alpha subunits of heterotrimeric Gproteins (RefSeq, 2002). RGS4 revealed a statistically significantdown-regulation in liver metastases and at the tumor invasion frontcompared with the primary pancreatic tumor (Niedergethmann et al.,2007). RGS4 is over-expressed very commonly in thyroid carcinoma, thoughit is not expressed in normal human tissues (Nikolova et al., 2008).RGS4 transcript was detected in non-cancerous immortalized ovariansurface epithelial cells at levels several thousand fold higher than itsexpression level in ovarian cancer cell lines (Hurst et al., 2009).

The gene for RPS6 encodes ribosomal protein S6, a cytoplasmic ribosomalprotein that is a component of the 40S subunit of ribosomes. RPS6 maycontribute to the control of cell growth and proliferation through theselective translation of particular classes of mRNA (RefSeq, 2002). RPS6is a downstream target of mTOR and has been found to be associated withmultiple physiological and pathophysiological functions (Chen et al.,2014a). RPS6 phosphorylation attenuates DNA damage and tumor suppressionduring development of pancreatic cancer (Khalaileh et al., 2013).

The gene for RPS8 encodes ribosomal protein S8, a cytoplasmic ribosomalprotein that is a component of the 40S subunit of ribosomes. RPS8expression is increased in colorectal tumors and colon polyps comparedto matched normal colonic mucosa (RefSeq, 2002). RPS8 up-regulation inpancreatic ductal adenocarcinoma patients is correlated with short-termsurvival (Chen et al., 2015).

The gene for RPS8P10 encodes ribosomal protein S8 pseudogene 10, apseudogene located on chromosome 15q11.2 (RefSeq, 2002).

The gene for SCG5 encodes secretogranin V (7B2 protein), aneuroendocrine secretory protein (Portela-Gomes et al., 2008). Aduplication spanning the 3′ end of the SCG5 gene and a region upstreamof the GREM1 locus may increase the risk of developing colorectal cancer(Jaeger et al., 2012; Yang et al., 2014b).

The gene for SERPINB2 encodes serpin peptidase inhibitor, Glade B(ovalbumin), member 2, an inhibitor of extracellular protease urokinaseplasminogen activator and tissue plasminogen activator (Schroder et al.,2014). SERPINB2 is expressed in a number of different tumors. SERPINB2expression is associated with favorable prognosis in breast andpancreatic cancers, but poor prognosis in endometrial, ovarian, andcolorectal cancers (Schroder et al., 2014).

The gene for SERPINB3 encodes the protease inhibitor serpin peptidaseinhibitor, Glade B (ovalbumin), member 3 (RefSeq, 2002). SERPINB3 is aRas-responsive factor that plays an important role in Ras-associatedcytokine production and tumorigenesis (Catanzaro et al., 2014). SERPINB3expression is up-regulated in hepatocellular carcinoma (Pontisso, 2014).SERPINB3 is associated with the development of ovarian cancer (Lim andSong, 2013).

The gene for SERPINB4 encodes the protease inhibitor serpin peptidaseinhibitor, Glade B (ovalbumin), member 4 (RefSeq, 2002). SERPINB4 is aRas-responsive factor that plays an important role in Ras-associatedcytokine production and tumorigenesis (Catanzaro et al., 2014). SERPINB4expression is up-regulated in hepatocellular carcinoma (Pontisso, 2014).

The gene for SERPINH1 encodes serpin peptidase inhibitor, clade H (heatshock protein 47), member 1, (collagen binding protein 1), a serineproteinase inhibitor. SERPINH1 functions as a collagen-specificmolecular chaperone in the endoplasmic reticulum (RefSeq, 2002).SERPINH1 is over-expressed in many human cancers, including stomachcancer, lung cancer, pancreatic ductal adenocarcinoma, glioma, andulcerative colitis-associated carcinomas (Zhao et al., 2014).

The gene for SEZ6L encodes seizure related 6 homolog (mouse)-like, atransmembrane protein with multiple domains involved in protein-proteininteraction and signal transduction (Nishioka et al., 2000). SEZ6L ishypermethylated in gastric cancer (Kang et al., 2008). SEZ6L expressionis up-regulated in non-small cell lung cancer and small cell lung cancercell lines as well as in primary tumor samples compared to normal lungtissues (Gorlov et al., 2007).

The gene for SLC16A3 encodes solute carrier family 16 member 3, aproton-linked monocarboxylate transporter (RefSeq, 2002). Most solidtumors are known to rely on glycolysis for energy production. High ratesof glycolysis result in an increased production of lactate which hasbeen associated with poor clinical outcome and direct contribution totumor growth and progression. SLC16A3 is one of few monocarboxylatetransporters which facilitate the lactate export in cancer cells (Dhupet al., 2012; Draoui and Feron, 2011). The SLC16A3 expression has beenassociated with poor prognosis in hepatocellular cancer patients andincreased cell proliferation, migration and invasion in cell lineexperiments (Gao et al., 2014a). The functional involvement of SLC16A3in the tumorigenesis was shown in a subset of pancreatic cancer (Baek etal., 2014).

The gene for MTCL1 encodes microtubule crosslinking factor 1. MTCL1 wasshown to be involved in the polarity-dependent microtubule remodelingand to mediate the epithelial-cell-specific reorganization ofnon-centrosomal microtubules through its microtubule-crosslinkingactivity (Sato et al., 2013).

The gene for SST encodes the pre-pro-protein of the hormonesomatostatin. Somatostatin is expressed throughout the body and inhibitsthe release of numerous secondary hormones. This hormone is an importantregulator of the endocrine system through its interactions withpituitary growth hormone, thyroid stimulating hormone, and most hormonesof the gastrointestinal tract. Somatostatin also affects rates ofneurotransmission in the central nervous system and proliferation ofboth normal and tumorigenic cells (RefSeq, 2002). SST analogues aresuccessfully used and further investigated as a therapeutic approach inthe treatment of gastroenteropancreatic neuroendocrine (carcinoid)tumors, hepatocellular cancer and breast cancer (Pivonello et al., 2014;Culler, 2011; Appetecchia and Baldelli, 2010; Modlin et al., 2010; Wattet al., 2008).

The gene for THY1 is a candidate tumor suppressor gene in nasopharyngealcarcinoma bearing anti-invasive activity (Lung et al., 2010).

The gene for TSC22D4 encodes a protein which is a member of the TSC22domain family of leucine zipper transcriptional regulators (RefSeq,2002). Hepatic levels of TSC22D4 were increased in cancer cachexia(Jones et al., 2013).

The gene for TUBA1A encodes tubulin, alpha 1a. The expression of TUBA1Ais predominantly found in morphologically differentiated neurologiccells. Mutations in this gene cause lissencephaly type 3 (LIS3)—aneurological condition characterized by microcephaly, mentalretardation, and early-onset epilepsy and caused by defective neuronalmigration (RefSeq, 2002). De-regulated expression of TUBA1A and someother genes, caused by chromosomal rearrangements inradiation-transformed and tumorigenic breast cell lines, might reflectearly molecular events in breast carcinogenesis (Unger et al., 2010).Using comparative proteomic analysis of advanced serous epithelialovarian carcinoma, TUBA1A was identified as one potential predictor forchemoresistance (Kim et al., 2011).

The gene for TUBA1B encodes tubulin, alpha 1 b (RefSeq, 2002). Thedifferential expression of TUBA1B in combination with the expression ofsome other genes was associated with prognosis in mantle cell lymphoma,prediction of relapse among patients with stage II colorectal cancer anddifferentiation between uveal melanomas that subsequently metastasizedand those that did not (Blenk et al., 2008; Agesen et al., 2012; Lingeet al., 2012). TUBA1B expression was up-regulated in hepatocellularcancer tissues and proliferating hepatocellular cancer cells. Anincreased TUBA1B expression was associated with poor overall survivaland resistance to paclitaxel of hepatocellular cancer patients (Lu etal., 2013a). In ovarian cancer cells, the reduced expression of TUBA1Bwas associated with oxaliplatin resistance (Tummala et al., 2009).

The gene for TUBA1C encodes tubulin, alpha 1c (RefSeq, 2002). Theexpression of TUBA1C was shown to be up-regulated in osteosarcoma andHCV-associated hepatocellular cancer and may be a potential biomarkerfor osteosarcoma tumorigenesis or well-differentiated HCV-associatedhepatocellular cancer (Kuramitsu et al., 2011; Li et al., 2010).

The gene for TUBA3C encodes tubulin, alpha 3c (RefSeq, 2002). The genefor TUBA3D encodes tubulin, alpha 3d (RefSeq, 2002). The gene for TUBA4Aencodes tubulin, alpha 4a (RefSeq, 2002). The comparative proteomicanalysis of esophageal squamous cell carcinoma (ESCC) showed anincreased expression of TUBA4A (Qi et al., 2005).

The gene for TUBA8 encodes tubulin, alpha 8. Mutations in TUBA8 areassociated with polymicrogyria and optic nerve hypoplasia (RefSeq,2002). In mouse liver, TUBA8 was induced after treatment withphenobarbital, a non-genotoxic carcinogen. In hepatocellular carcinomacell lines, the over-expression of TUBA8 was shown to affect cellgrowth, proliferation and migration (Kamino et al., 2011).

The gene for UCN3 is a member of the sauvagine/corticotropin-releasingfactor/urotensin I family. It is structurally related to thecorticotropin-releasing factor (CRF) gene and the encoded product is anendogenous ligand for CRF type 2 receptors. In the brain it may beresponsible for the effects of stress on appetite (RefSeq, 2002). Ucn3is produced in normal adrenal and adrenal tumors (both adrenocorticaltumors and pheochromocytomas), and acts as an autocrine or paracrineregulator in normal adrenal and adrenal tumors (Takahashi et al., 2006).Urocortin 3 activates AMPK and AKT pathways and enhances glucosedisposal in rat skeletal muscle (Roustit et al., 2014).

The gene for VCAN is a member of the aggrecan/versican proteoglycanfamily. The encoded protein is a large chondroitin sulfate proteoglycanand is a major component of the extracellular matrix. This protein isinvolved in cell adhesion, proliferation, migration and angiogenesis andplays a central role in tissue morphogenesis and maintenance (RefSeq,2002). VCAN expression was regulated in cancer-associated fibroblaststhrough TGF-beta receptor type II and SMAD signaling. up-regulated VCANpromoted the motility and invasion of ovarian cancer cells by activatingthe NF-kappaB signaling pathway and by up-regulating expression of CD44,matrix metalloproteinase-9, and the hyaluronan-mediated motilityreceptor (Yeung et al., 2013). A collagen-remodeling gene signatureincluding VCAN regulated by TGF-beta signaling is associated withmetastasis and poor survival in serous ovarian cancer (Cheon et al.,2014). VCAN is significantly up-regulated in CRC comparing pairedsamples of healthy colon mucosa and tumor tissues of 53 patients (Pituleet al., 2013).

The gene for WNT16, wingless-type MMTV integration site family, member16 encodes a secreted signaling protein which is implicated inoncogenesis and in several developmental processes, including regulationof cell fate and patterning during embryogenesis (RefSeq, 2002). Theexpression of WNT16 was shown to be up-regulated in t (1;19) chromosomaltranslocation-containing acute lymphoblastoid leukemia (ALL) and play animportant role in leukemogenesis (Casagrande et al., 2006; Mazieres etal., 2005). A study of ALL cell lines and samples from patients with ALLshowed that the up-regulation of WNT16 and few other Wnt target geneswas caused by the methylation of Wnt inhibitors which was furtherassociated with significantly decreased 10-year disease-free survivaland overall survival (Roman-Gomez et al., 2007).

The gene for WNT5A belongs to the WNT gene family that consists ofstructurally related genes which encode secreted signaling proteins.These proteins have been implicated in oncogenesis and in severaldevelopmental processes, including regulation of cell fate andpatterning during embryogenesis. The WNT5A gene encodes a member of theWNT family that signals through both the canonical and non-canonical WNTpathways. This protein is a ligand for the seven transmembrane receptorfrizzled-5 and the tyrosine kinase orphan receptor 2. This protein playsan essential role in regulating developmental pathways duringembryogenesis. This protein may also play a role in oncogenesis (RefSeq,2002). WNT5A is over-expressed in CRC and had a concordance rate of 76%between the primary tumor and metastatic site (Lee et al., 2014). WNT5Ais up-regulated and a key regulator of the epithelial-to-mesenchymaltransition and metastasis in human gastric carcinoma cells,nasopharyngeal carcinoma and pancreatic cancer (Kanzawa et al., 2013;Zhu et al., 2014; Bo et al., 2013).

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, 13 or 14 or longer,and in case of MHC class II peptides (elongated variants of the peptidesof the invention) they can be as long as 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid (s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 67 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 67, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 67. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 67, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or—II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acids whose incorporation do not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according toSEQ ID NO: 4, 29, and 30 Position 1 2 3 4 5 6 7 8 9 SEQ ID NO. 4 S V D VS P P K V Variants I L A L I L L L L A A I A L A A A M I M L M M A T I TL T T A Q I Q L Q Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID NO: 29 F L Q E YL D A I Variants I L I V I I A M L M V M M A A L A V A A A V L V V V V AT L T V T T A Q L Q V Q Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID NO. 30 V VD E G P T G V Variants L I A M L M I M M A A L A I A A A V L V I V V A TL T I T T A Q L Q I Q Q A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid (s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 67.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 67 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly (ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, (Bruckdorfer et al., 2004), and the references ascited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of natural TUMAPsrecorded from pancreatic cancer samples (N=18 A*02-positive samples)with the fragmentation patterns of corresponding synthetic referencepeptides of identical sequences. Since the peptides were directlyidentified as ligands of HLA molecules of primary tumors, these resultsprovide direct evidence for the natural processing and presentation ofthe identified peptides on primary cancer tissue obtained from 18pancreatic cancer patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from pancreatic cancer tissue samples werepurified and HLA-associated peptides were isolated and analyzed by LC-MS(see examples). All TUMAPs contained in the present application wereidentified with this approach on primary pancreatic cancer samplesconfirming their presentation on primary pancreatic cancer.

TUMAPs identified on multiple pancreatic cancer and normal tissues werequantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably pancreatic cancer that over- or exclusivelypresent the peptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanpancreatic cancer samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy pancreas cells or other normal tissue cells, demonstrating ahigh degree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from pancreatic cancer, but not on normaltissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. pancreatic cancer cells presenting the derivedpeptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutics such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 67, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. The peptide(s) may be derived from one or more specific TAAs and may bind to MHCclass I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasm ids pRS413-416 are Yeast Centromere plasmids(Ycps). CMV promoter-based vectors (for example from Sigma-Aldrich)provide transient or stable expression, cytoplasmic expression orsecretion, and N-terminal or C-terminal tagging in various combinationsof FLAG, 3×FLAG, c-myc or MAT. These fusion proteins allow fordetection, purification and analysis of recombinant protein. Dual-taggedfusions provide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide (s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such as Poly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly (IC-R), poly (I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex (es). Binding to other peptide-MHC complexes is irrelevant ifthe peptide of the cross-reactive peptide-MHC is not naturallyoccurring, i.e. not derived from the human HLA-peptidome. Tests toassess target cell killing are well known in the art. They should beperformed using target cells (primary cells or cell lines) withunaltered peptide-MHC presentation, or cells loaded with peptides suchthat naturally occurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 67,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 67, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 67 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:67 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 67, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 67.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of pancreatic cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 67 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are pancreatic cancer cells orother solid or hematological tumor cells such as pancreatic cancer,brain cancer, kidney cancer, colon or rectal cancer, leukemia.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of pancreatic cancer. The present invention also relates tothe use of these novel targets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a pancreatic cancer marker(poly)peptide, delivery of a toxin to a pancreatic cancer cellexpressing a cancer marker gene at an increased level, and/or inhibitingthe activity of a pancreatic cancer marker polypeptide) according to theinvention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length pancreatic cancer marker polypeptides orfragments thereof may be used to generate the antibodies of theinvention. A polypeptide to be used for generating an antibody of theinvention may be partially or fully purified from a natural source, ormay be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 67polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the pancreatic cancer markerpolypeptide used to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain (s) is identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F (ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating pancreaticcancer, the efficacy of the therapeutic antibody can be assessed invarious ways well known to the skilled practitioner. For instance, thesize, number, and/or distribution of cancer in a subject receivingtreatment may be monitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 67, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively eliciting high-or low-avidity antigen-specific T cell responses with high efficiencyfrom blood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells.

In addition plant viruses may be used (see, for example, Porta et al.(Porta et al., 1994) which describes the development of cowpea mosaicvirus as a high-yielding system for the presentation of foreignpeptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 67.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule (s) of the invention or (a) known molecule(s).

The present invention further provides a medicament that is useful intreating cancer, in particular pancreatic cancer and other malignancies.

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from pancreaticcancer, the medicament of the invention is preferably used to treatpancreatic cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue ofpancreatic cancer patients with various HLA-A HLA-B and HLA-C alleles.It may contain MHC class I and MHC class II peptides or elongated MHCclass I peptides. In addition to the tumor associated peptides collectedfrom several pancreatic cancer tissues, the warehouse may containHLA-A*02 and HLA-A*24 marker peptides. These peptides allow comparisonof the magnitude of T-cell immunity induced by TUMAPS in a quantitativemanner and hence allow important conclusion to be drawn on the capacityof the vaccine to elicit anti-tumor responses. Secondly, they functionas important positive control peptides derived from a “non-self” antigenin the case that any vaccine-induced T-cell responses to TUMAPs derivedfrom “self” antigens in a patient are not observed. And thirdly, it mayallow conclusions to be drawn, regarding the status of immunocompetenceof the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, pancreatic cancer samples frompatients and blood from healthy donors were analyzed in a stepwiseapproach:

1. HLA ligands from the malignant material were identified by massspectrometry2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue(pancreatic cancer) compared with a range of normal organs and tissues3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as frompancreatic cancer patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients tumor and, wherepossible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from pancreatic cancer cells and since it was determinedthat these peptides are not or at lower levels present in normaltissues, these peptides can be used to diagnose the presence of acancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for pancreatic cancer. Presence of groups of peptides canenable classification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying Figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGS. 1A-1C shows the over-presentation of various peptides in normaltissues (dark gray) and pancreatic cancer (light gray). FIG. 1D showsall cell lines (dark gray), normal tissues (gray) and cancers tissues(light gray) where the exemplary peptide (FLFDGSANLV) (SEQ ID NO.: 9)has been detected. FIG. 1A) Gene: CTLA/CTLB, Peptide: FLAQQESEI (A*02)(SEQ ID NO.: 1); Tissues shown from left to right: 1 adipose tissues, 3adrenal glands, 2 arteries, 3 bone marrows, 7 brains, 3 breasts, 13colons, 1 ovary, 4 esophagi, 2 gallbladders, 3 hearts, 12 kidneys, 4leukocyte samples, 19 livers, 43 lungs, 1 lymph node, 1 ovary, 2peripheral nerves, 1 peritoneum, 1 pituitary gland, 3 pleuras, 1prostate, 6 recti, 3 skeletal muscles, 3 skins, 2 small intestines, 4spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2veins, 6 pancreas, 18 pancreatic cancers; FIG. 1B) Gene: PLEC, Peptide:SLQEEHVAVA (A*02), (SEQ ID NO.: 2); Tissues shown from left to right: 1adipose tissues, 3 adrenal glands, 2 arteries, 3 bone marrows, 7 brains,3 breasts, 13 colons, 1 ovary, 4 esophagi, 2 gallbladders, 3 hearts, 12kidneys, 4 leukocyte samples, 19 livers, 43 lungs, 1 lymph node, 1ovary, 2 peripheral nerves, 1 peritoneum, 1 pituitary gland, 3 pleuras,1 prostate, 6 recti, 3 skeletal muscles, 3 skins, 2 small intestines, 4spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroid glands, 2 uteri, 2veins, 6 pancreas, 18 pancreatic cancers; FIG. 1C) Gene: COL6A3,Peptide: FLVDGSSAL (A*02) (SEQ ID NO.: 10); Tissues shown from left toright: 1 adipose tissues, 3 adrenal glands, 2 arteries, 3 bone marrows,7 brains, 3 breasts, 13 colons, 1 ovary, 4 esophagi, 2 gallbladders, 3hearts, 12 kidneys, 4 leukocyte samples, 19 livers, 43 lungs, 1 lymphnode, 1 ovary, 2 peripheral nerves, 1 peritoneum, 1 pituitary gland, 3pleuras, 1 prostate, 6 recti, 3 skeletal muscles, 3 skins, 2 smallintestines, 4 spleens, 5 stomachs, 1 testis, 2 thymi, 3 thyroid glands,2 uteri, 2 veins, 6 pancreas, 18 pancreatic cancers; FIG. 1D) COL6A3,Peptide: FLFDGSANLV (A*02) (SEQ ID NO.: 9); Tissues shown from left toright: 5 pancreatic cancer cell lines, 7 normal tissues (1 colon, 6lungs), 85 cancer tissues (2 breast cancers, 6 colon cancers, 4esophageal cancers, 3 liver cancers, 56 lung cancers, 5 pancreaticcancers, 3 rectal cancers, 1 melanoma, 5 gastric cancers). The set ofnormal tissues was the same as in A-C, but tissues without detection arenot shown. Discrepancies regarding the list of tumor types between FIG.1D and table 4 might be due to the more stringent selection criteriaapplied in table 4 (for details please refer to table 4). FIG. 1D showsall samples with detectable presentation of the peptide Y, regardless ofover-presentation parameters and technical sample quality check.

FIGS. 1E-1I show all cell lines (dark gray), normal tissues (gray) andcancers tissues (light gray) where the exemplary peptides have beendetected. FIG. 1E) Peptide: SVDVSPPKV (A*02) (SEQ ID NO.: 4); Tissuesshown from left to right: 1 cell-lines, 3 primary cultures, 1 skin, 1bile duct cancer, 3 brain cancers, 1 breast cancer, 4 esophagealcancers, 5 kidney cancers, 11 lung cancers, 1 lymph node cancer, 1ovarian cancer, 3 pancreas cancers, 1 prostate cancer, 3 skin cancers, 2urinary bladder cancers, 3 uterus cancers; FIG. 1F) Peptide: LLVDDSFLHTV(A*02) (SEQ ID NO.: 5); Tissues shown from left to right: 2 cell-lines,1 primary culture, 1 bile duct cancer, 2 brain cancers, 1 breast cancer,3 esophageal cancers, 2 gallbladder cancers, 2 kidney cancers, 2 livercancers, 3 lung cancers, 7 ovarian cancers, 2 pancreas cancers, 3 skincancers, 1 stomach cancer, 1 uterus cancer, FIG. 1G) Peptide: IVDDLTINL(A*02) (SEQ ID NO.: 8); Tissues shown from left to right: 1 cell-line, 1colon cancer, 2 esophageal cancers, 2 gallbladder cancers, 5 lungcancers, 1 lymph node cancer, 1 pancreas cancer, 2 skin cancers, 4stomach cancers, 1 urinary bladder cancer, 4 uterus cancers, FIG. 1H)Peptide: LLAGQTYHV (A*02) (SEQ ID NO.: 13); Tissues shown from left toright: 6 cell-lines, 1 lung, 1 placenta, 2 bile duct cancers, 3 breastcancers, 2 colon cancers, 2 esophageal cancers, 2 gallbladder cancers, 1liver cancer, 36 lung cancers, 3 ovarian cancers, 3 pancreas cancers, 1rectum cancer, 3 urinary bladder cancers; and FIG. 1I) Peptide:VLAKPGVISV (A*02) (SEQ ID NO.: 14); Tissues shown from left to right: 7cell-lines, 1 lung, 1 bile duct cancer, 4 breast cancers, 1 coloncancer, 2 esophageal cancers, 1 gallbladder cancer, 36 lung cancers, 1ovarian cancer, 3 pancreas cancers, 2 rectum cancers, 1 stomach cancer,1 urinary bladder cancer.

FIGS. 2A-2C shows exemplary expression profiles (relative expressioncompared to normal kidney) of source genes of the present invention thatare highly over-expressed or exclusively expressed in pancreatic cancerin a panel of normal tissues (dark gray) and 11 pancreatic cancersamples (gray). FIG. 2A) LAMC2; Tissues from left to right: 1 adrenalgland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1esophagus, 1 heart, 3 kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1lymph node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivarygland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 testis, 1 thymus, 1 thyroid gland, 1 urinary bladder, 1uterine cervix, 1 uterus, 1 vein, 18 pancreatic cancers; FIG. 2B) VCAN;Tissues from left to right: 1 adrenal gland, 1 artery, 1 bone marrow, 1brain (whole), 1 breast, 1 colon, 1 esophagus, 1 heart, 3 kidneys, 1leukocyte sample, 1 liver, 1 lung, 1 lymph node, 1 ovary, 1 pancreas, 1placenta, 1 prostate, 1 salivary gland, 1 skeletal muscle, 1 skin, 1small intestine, 1 spleen, 1 stomach, 1 testis, 1 thymus, 1 thyroidgland, 1 urinary bladder, 1 uterine cervix, 1 uterus, 1 vein, 18pancreatic cancers; FIG. 2C) FAP; Tissues from left to right: 1 adrenalgland, 1 artery, 1 bone marrow, 1 brain (whole), 1 breast, 1 colon, 1esophagus, 1 heart, 3 kidneys, 1 leukocyte sample, 1 liver, 1 lung, 1lymph node, 1 ovary, 1 pancreas, 1 placenta, 1 prostate, 1 salivarygland, 1 skeletal muscle, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 testis, 1 thymus, 1 thyroid gland, 1 urinary bladder, 1uterine cervix, 1 uterus, 1 vein, 18 pancreatic cancers.

FIGS. 3A-3D shows exemplary immunogenicity data: flow cytometry resultsafter peptide-specific multimer staining. FIGS. 3C and 3D show exemplaryresults of peptide-specific in vitro CD8+ T cell responses of a healthyHLA-A*02+ donor. CD8+ T cells were primed using artificial APCs coatedwith anti-CD28 mAb and HLA-A*02 in complex with Seq ID No 3 peptide(FIG. 3C, left panel) or Seq ID No 50 peptide (FIG. 3D, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*02/Seq ID No 3 (FIG. 3C) or A*02/Seq ID No 50 (FIG. 3D). Right panels(FIGS. 3C and 3D) show control staining of cells stimulated withirrelevant A*02/peptide complexes. Viable singlet cells were gated forCD8+ lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

EXAMPLES Example 1: Identification and Quantitation of Tumor AssociatedPeptides Presented on the Cell Surface Tissue Samples

Patients' tumor tissues were obtained from Asterand (Detroit, USA andRoyston, Herts, UK); Geneticist Inc. (Glendale, Calif., USA); Hospitalof Heidelberg; University Hospital of Tübingen. Normal tissues wereobtained from Bio-Options Inc. (CA, USA); BioServe (Beltsville, Md.,USA); Capital BioScience Inc. (Rockville, Md., USA); Geneticist Inc.(Glendale, Calif., USA); University Hospital of Geneva; UniversityHospital of Heidelberg; Kyoto Prefectural University of Medicine (KPUM);University Hospital Munich; ProteoGenex Inc. (Culver City, Calif., USA);University Hospital of Tübingen. Written informed consents of allpatients had been given before surgery or autopsy. Tissues wereshock-frozen immediately after excision and stored until isolation ofTUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose pancreatic cancer samples to a baseline of normaltissue samples. Presentation profiles of exemplary over-presentedpeptides are shown in FIGS. 2A-2C. Presentation scores for exemplarypeptides are shown in Table 8.

TABLE 8 Presentation scores. The table lists peptides thatare very highly over-presented on tumors comparedto a panel of normal tissues (+++), highly over-presented on tumors compared to a panel of normaltissues (++) or over-presented on tumors comparedto a panel of normal tissues (+). SEQ ID Peptide No. SequencePresentation  1 FLAQQESEI +++  2 SLQEEHVAVA ++  3 ALLTFMEQV +++  4SVDVSPPKV +  5 LLVDDSFLHTV +++  7 AQQESEIAGI +++  8 IVDDLTINL +++  9FLFDGSANLV +++ 10 FLVDGSSAL +++ 11 FLYKIIDEL +++ 12 FVSEIVDTV +++ 13LLAGQTYHV ++ 14 VLAKPGVISV + 15 SLANNVTSV + 16 APVNVTTEVKSV +++ 17FLKSGDAAIV +++ 18 SLLDDELMSL ++ 19 HLAPETDEDDL +++ 20 RLAGDGVGAV ++ 21HLMDQPLSV +++ 23 SLSAFTLFL + 24 GLLEELVTV +++ 25 SLKEEVGEEAI + 26SLKEEVGEEAIV ++ 29 FLQEYLDAI +++ 31 SLAAAAGKQEL +++ 32 SLAAAAGKQELA +++33 SLDSRLELA +++ 34 MLMPVHFLL +++ 35 VMDSGDGVTHTV + 36 KQEYDESGPSIVH +++37 GLLKKINSV +++ 38 NLVEKTPALV +++ 39 TLLSNLEEA + 40 FILDSAETTTL +++ 41FLLDGSEGV +++ 42 KLVDKSTEL +++ 43 RLDQRVPQI ++ 46 TFAPVNVTTEVKSV + 47KMDASLGNLFA +++ 48 ALTQTGGPHV +++ 49 NLKGTFATL +++ 50 ALAAILTRL +++ 51ALMLQGVDL +++ 52 RMVEEIGVEL ++ 56 GLLDYATGAIGSV +++ 57 FLGKVVIDV +++ 58GLAAFKAFL +++ 59 KLFNLSKEDDV +++ 61 ALEKDYEEVGV +++ 62 ALEKDYEEV +++ 63FAGDDAPR +++ 64 FLVSNMLLAEA +++ 66 ALLSGLREA +++ 67 KMFFLIDKV +++ 68KLLTEVHAA +++ 70 FLVDGSWSV +++ 71 FLLDGSANV +++ 74 KIQEILTQV +++ 75RLDDLKMTV ++ 76 RLLDSVSRL + 77 GLTDNIHLV +++ 79 VLAPRVLRA + 80TLYPHTSQV + 81 AMSSKFFLV +++ 82 SISDVIAQV +++ 83 FLIDSSEGV +++ 84NLLDLDYEL +++ 85 TVAEVIQSV ++ 86 SLLAQNTSWLL ++ 87 LLLGSPAAA +++

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalization, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0. Exemplary expression profiles of source genes of the presentinvention that are highly over-expressed or exclusively expressed inpancreatic cancer are shown in FIGS. 1A-1I. Expression scores forfurther exemplary genes are shown in Table 9.

TABLE 9 Expression scores. The table lists peptides fromgenes that are very highly overexpressed in tumorscompared to a panel of normal tissues (+++),highlyoverexpressed in tumors compared to a panel ofnormal tissues (++) or overexpressed in tumorscompared to a panel of normal tissues (+). Gene SEQ ID No SequenceExpression  3 ALLTFMEQV ++  4 SVDVSPPKV +  6 VLISLKQAPLV + 13LLAGQTYHV + 15 SLANNVTSV + 16 APVNVTTEVKSV + 20 RLAGDGVGAV + 23SLSAFTLFL + 25 SLKEEVGEEAI ++ 27 YLQGQRLDNV + 30 VVDEGPTGV ++ 36KQEYDESGPSIVH + 43 RLDQRVPQI + 44 VLLDKIKNLQV + 46 TFAPVNVTTEVKSV ++ 47KMDASLGNLFA + 48 ALTQTGGPHV + 50 ALAAILTRL +++ 51 ALMLQGVDL ++ 52RMVEEIGVEL + 57 FLGKVVIDV + 58 GLAAFKAFL + 59 KLFNLSKEDDV + 61ALEKDYEEVGV +++ 62 ALEKDYEEV +++ 66 ALLSGLREA ++ 67 KMFFLIDKV + 71FLLDGSANV + 73 TLVAIVVGV ++ 75 RLDDLKMTV ++ 76 RLLDSVSRL +++ 78TLSSIKVEV +++ 81 AMSSKFFLV ++

Example 3 In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for 22 HLA-A*0201 restricted TUMAPsof the invention so far, demonstrating that these peptides are T-cellepitopes against which CD8+ precursor T cells exist in humans (Table10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO 88) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI (SEQ ID No. 89) fromDDX5), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Pancreatic Cancer Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIGS. 3A-3D together withcorresponding negative controls. Results for 2 peptides from theinvention are summarized in Table 10.

TABLE 10 in vitro immunogenicity of HLA class I peptides of theinvention Exemplary results of in vitro immunogenicity experimentsconducted by the applicant for the peptides of the invention. Seq IDwells donors 69 ++ ++++ 87 + +++ <20% = +; 20%-49% = ++; 50%-69% =+++; >=70% = ++++Results for 7 additional peptides from the invention are summarized inTable 10B.

TABLE 10B in vitro immunogenicity of HLA class I peptidesof the invention. Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +; 20%-49% = ++; 50%-69% = +++; > =70% = ++++ Seq ID No Sequence Wells positive [%]  3 ALLTFMEQV ++ 20RLAGDGVGAV ++++ 21 HLMDQPLSV + 23 SLSAFTLFL ++ 34 MLMPVHFLL + 37GLLKKINSV + 50 ALAAILTRL +++

Example 4 Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizates (trifluoro acetate salt) in purities of >50%. All TUMAPsare preferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5 MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100 fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. <20% = +; 20%-49% = ++; 50%-75% =+++; > = 75% = +++; SEQ ID No Sequence Peptide exchange  1 FLAQQESEI ++ 2 SLQEEHVAVA ++  3 ALLTFMEQV +++  4 SVDVSPPKV ++  5 LLVDDSFLHTV +++  6VLISLKQAPLV ++  7 AQQESEIAGI ++  8 IVDDLTINL ++  9 FLFDGSANLV ++ 10FLVDGSSAL ++ 11 FLYKIIDEL +++ 12 FVSEIVDTV +++ 13 LLAGQTYHV ++ 14VLAKPGVISV ++ 15 SLANNVTSV ++ 16 APVNVTTEVKSV ++ 17 FLKSGDAAIV ++ 18SLLDDELMSL ++ 20 RLAGDGVGAV ++ 21 HLMDQPLSV ++ 22 TLDGAAVNQV ++ 23SLSAFTLFL ++ 24 GLLEELVTV ++ 25 SLKEEVGEEAI ++ 26 SLKEEVGEEAIV ++ 27YLQGQRLDNV ++ 28 YLQGQRLDNVV ++ 29 FLQEYLDAI +++ 30 VVDEGPTGV ++ 31SLAAAAGKQEL ++ 32 SLAAAAGKQELA + 33 SLDSRLELA ++ 34 MLMPVHFLL ++++ 35VMDSGDGVTHTV ++ 37 GLLKKINSV ++ 38 NLVEKTPALV +++ 39 TLLSNLEEA ++ 40FILDSAETTTL ++ 41 FLLDGSEGV +++ 42 KLVDKSTEL ++ 43 RLDQRVPQI ++ 44VLLDKIKNLQV ++ 46 TFAPVNVTTEVKSV ++ 47 KMDASLGNLFA ++++ 48 ALTQTGGPHV ++49 NLKGTFATL + 50 ALAAILTRL +++ 51 ALMLQGVDL ++ 52 RMVEEIGVEL ++ 53SSFGGLGGGSV + 54 VLLSEIEVA ++ 55 YLDAMMNEA ++ 56 GLLDYATGAIGSV +++ 57FLGKVVIDV ++++ 58 GLAAFKAFL +++ 59 KLFNLSKEDDV ++ 60 YLEEDVYQL ++ 64FLVSNMLLAEA +++ 65 YLYDSETKNA ++ 66 ALLSGLREA +++ 67 KMFFLIDKV +++

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1. A method of treating cancer in a HLA-A*02+ patient, wherein saidcancer comprises cancer cells that overexpress a polypeptide and presentat their surface in complex with an MHC class I molecule a peptideconsisting of the amino acid sequence of SEQ ID NO: 10, said methodcomprising administering to said patient an effective amount ofactivated antigen-specific CD8+ cytotoxic T cells to kill cancer cells,wherein said activated antigen-specific CD8+ cytotoxic T cells areproduced by contacting in vitro CD8+ cytotoxic T cells with an antigenpresenting cell presenting at its surface in complex with an MHC class Imolecule a peptide consisting of the amino acid sequence of SEQ ID NO:10, wherein said cancer is selected from pancreatic cancer, colon orrectal cancer, melanoma, esophageal cancer, breast cancer, small celllung cancer, stomach cancer, urinary bladder cancer, gall bladdercancer, and bile duct cancer.
 2. The method of claim 1, wherein thecytotoxic T cells produced by contacting CD8+ cytotoxic T cells withsaid antigen presenting cell are cytotoxic T cells autologous to thepatient.
 3. The method of claim 1, wherein the cytotoxic T cellsproduced by contacting CD8+ cytotoxic T cells with said antigenpresenting cell are cytotoxic T cells obtained from a healthy donor. 4.The method of claim 1, wherein the cytotoxic T cells produced bycontacting CD8+ cytotoxic T cells with said antigen presenting cell arecytotoxic T cells isolated from tumor infiltrating lymphocytes orperipheral blood mononuclear cells.
 5. The method of claim 1, whereinthe cytotoxic T cells produced by contacting CD8+ cytotoxic T cells withsaid antigen presenting cell are expanded in vitro before beingadministered to the patient.
 6. The method of claim 1, wherein thecytotoxic T cells are expanded in vitro in the presence of an anti-CD28antibody and IL-12.
 7. The method of claim 1, wherein the effectiveamount of activated antigen-specific CD8+ cytotoxic T cells to kill thecancer cells are administered in the form of a composition.
 8. Themethod of claim 7, wherein said composition further comprises anadjuvant.
 9. The method of claim 8, wherein said adjuvant is selectedfrom agonistic anti-CD40 antibody, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, interferon-alpha, interferon-beta, CpGoligonucleotides, poly-(I:C), RNA, sildenafil, particulate formulationswith poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 10. The methodof claim 8, wherein the adjuvant comprises IL-2.
 11. The method of claim8, wherein the adjuvant comprises IL-21.
 12. The method of claim 1,wherein the antigen presenting cell is a dendritic cell or a macrophage.13. The method of claim 1, wherein the antigen presenting cell isinfected with a recombinant virus expressing a peptide consisting of theamino acid sequence of SEQ ID NO:
 10. 14. The method of claim 1, whereinthe antigen presenting cell is an artificial antigen presenting cell(aAPC) comprising an anti-CD28 antibody coupled to its surface.
 15. Themethod of claim 1, wherein the cancer is pancreatic cancer.
 16. Themethod of claim 1, wherein the cancer is colon or rectal cancer.
 17. Themethod of claim 1, wherein the cancer is melanoma.
 18. The method ofclaim 1, wherein the cancer is breast cancer.
 19. The method of claim 1,wherein the cancer is small cell lung cancer.
 20. The method of claim 1,wherein the cancer is esophageal cancer.